CN1344322A - BASBO55 polynucleotide and polypeptide from neisseria meningitidis uses thereof - Google Patents

Abstract

The invention provides BASB055 polypeptides and polynucleotides encoding BASB055 polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are diagnostic, prophylactic and therapeutic uses.

Description

Neisseria meningitidis BASB055 polynucleotides and polypeptides and uses thereof

Field of Invention

The present invention relates to polynucleotides (herein referred to as "BASB055 polynucleotides"), their encoded polypeptides (herein referred to as "BASB055" or "BASB055 polypeptides"), recombinant substances and their preparation methods. In another aspect, the invention relates to methods of using such polypeptides and polynucleotides, including vaccines against bacterial infections. In yet another aspect, the invention relates to diagnostic assays for the detection of infection by specific pathogens.

Background of the Invention

Neisseria meningitidis (meningococcus) is a Gram-negative bacterium frequently isolated from the upper respiratory tract of humans. It can occasionally cause invasive bacterial diseases such as bacteremia and meningitis. The incidence of meningitic disease shows geographic, seasonal and annual variations (Schwartz, B. Moore, P.S., Broome, C.V.; Clinical Microbiology Reviews 2(Suppl), S18-S24, 1989). In temperate countries, where most disease is caused by serotype B strains, the annual incidence varies between 1 to 10 per 100,000 population, sometimes reaching higher values (Kaczmarski, E.B. (1997 ), Commun.Dis.Rep.Rev.7: R55-9, 1995; Scholten, R.J.P.M., Bijlmer, H.A., Poolman, J.T. et al. "Clinical Infectious Diseases" 16:237-246, 1993; Cruz, C., Pavez , G., Aguilar, E., et al. Infection Epidemiology 105: 119-126, 1990).

Epidemics caused by serotype A meningococci, mainly in central Africa, sometimes reach 1,000 per 100,000 per year (Schwartz, B., Moore, P.S., Broome, C.V. Clinical Microbiology Reviews 2(suppl), S18-S24, 1989). Looking at meningitis disease as a whole, almost all cases are caused by meningococci serotypes A, B, C, W-135, and Y, and a tetravalent A, C, W-135, Polysaccharide vaccine of Y (Armand, J., Arminjon, F., Mynard, M.C., Lafaix, C., "J. Biol. Stand" 10:335-339, 1982). Polysaccharide vaccines are currently being improved by chemically coupling them to carrier proteins (Lieberman, J.M., Chiu, S.S., Wong, V.K. et al., JAMA 275:1499-1503, 1996).

A vaccine for serotype B has not been obtained because the capsular polysaccharide of B was found to be non-immunogenic, likely due to its structural similarity to host components (Wyle, F.A., Artenstein, M.S., Brandt, M.L. et al., Journal of Infectious Diseases 126:514-522, 1972; Finne, J.M., Leinonen, M., Mkel, P.M. The Lancet ii:355-357, 1983).

Efforts have been made for many years to develop vaccines based on the meningococcal outer membrane (deMoraes, J.C., Perkins, B., Camargo, M.C. et al. The Lancet 340:1074-1078, 1992; Bjune, G., Hoiby, E.A. Gronnesby, J.K. et al., 338:1093-1096, 1991). This vaccine has demonstrated efficacy in older children (>4 years) and adults ranging from 57% to 85%.

Many bacterial outer membrane components, such as PorA, PorB, Rmp, Opc, Opa, FrpB, are present in these vaccines and their contribution to the observed protection remains to be determined. Other bacterial outer membrane components that may be involved in the induction of protective immunity, such as TbpB and NspA, have been identified using animal or human antibodies (Martin, D., Cadieux, N., Hamel, J., Brodeux, B.R., Experimental Medical Journal 185: 1173-1183, 1997; Lissolo, L., Maitre-Wilmotte, C., Dumas, P. et al., Inf. Immun. 63: 884-890, 1995). The mechanism of protective immunity would involve antibody-mediated bactericidal activity and phagocytosis.

A bacteremia animal model has been used to combine all antibody-mediated mechanisms (Saukkonen, K., Leinonen, M., Abdillahi, H. Poolman, J.T. Vaccines 7:325-328, 1989). Bactericidal mechanisms mediated by late complement components are generally believed to be critical for immunity against meningitic disease (Ross, S.C., Rosenthal P.J., Berberic, H.M., Densen, P.J. J. Infectious Diseases 155:1266-1275, 1987).

The incidence of Neisseria meningitidis has increased considerably over the past few decades. This has been blamed on the emergence of multi-antibiotic resistant strains and an increase in populations of people with weakened immune systems. It is no longer unusual to isolate strains that are resistant to some or all standard antibiotics. This phenomenon has created an insatiable need for new antimicrobial agents, vaccines, drug screening methods and diagnostic tests against this microorganism.

            Invention Brief

The present invention relates to BASB055, especially BASB055 polypeptide and BASB055 polynucleotide, recombinant substances and their preparation methods. In another aspect, the invention relates to methods of using such polypeptides and polynucleotides, including the prevention and treatment of microbial diseases and others. In a further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and symptoms associated with such infections, such as assays for detecting the expression or activity of BASB055 polynucleotides or polypeptides.

Various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from reading the following description and other parts of the present disclosure.

                    Invention Details

As described in detail below, the present invention relates to BASB055 polypeptides and polynucleotides. Specifically, the present invention relates to polypeptides and polynucleotides of BASB055 of Neisseria meningitidis, and BASB055 has homology in amino acid sequence with MtrC protein of Neisseria gonorrhoeae. The present invention particularly relates to BASB055 having the nucleotide and amino acid sequences listed in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. It should be understood that the sequences listed as "DNA" in the following Sequence Listing represent an illustration of one embodiment of the present invention, since those of ordinary skill know that such sequences are commonly used as polynucleotides, including polyribonucleotides . polypeptide

In one aspect of the invention there are provided polypeptides of Neisseria meningitidis, referred to herein as "BASB055" and "BASB055 polypeptide", and biologically, diagnostic, prophylactic, clinically or therapeutically useful variants thereof , and compositions containing them. The present invention further provides: (a) an isolated polypeptide comprising an amino acid sequence having at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity to the amino acid sequence of SEQ ID NO: 2 % identity, most preferably at least 97-99% identity or complete identity; (b) a polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence that is identical to SEQ ID NO : 1 is at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, more preferably at least 97-99% identical or completely identical over its entire length; A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide having at least 85% identity to the amino acid sequence of SEQ ID NO: 2, more preferably At least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% identity or complete identity;

The BASB055 polypeptide provided in SEQ ID NO: 2 is the BASB055 polypeptide from Neisseria meningitidis strain ATCC13090.

The present invention also provides an immunogenic fragment of the BASB055 polypeptide, that is, a continuous part of the BASB055 polypeptide, which has the same or substantially the same immunogenic activity as the polypeptide comprising the amino acid sequence of SEQ ID NO:2. That is, the fragment (coupled with a carrier if necessary) is capable of generating an immune response capable of recognizing the BASB055 polypeptide. Such immunogenic fragments may include, for example, BASB055 polypeptides lacking the N-terminal leader sequence and/or the transmembrane region and/or the C-terminal anchor region. In a preferred aspect, the immunogenic fragment of BASB055 according to the present invention comprises substantially all extracellular domains of a polypeptide, wherein the polypeptide has at least 100% of the total length of SEQ ID NO:2 with SEQ ID NO:2 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% identity.

A fragment is a polypeptide that has an amino acid sequence that is partially, but not completely, identical to any amino acid sequence of any polypeptide of the invention. With respect to the BASB055 polypeptide, fragments may be "stand alone", or within a larger polypeptide, constituting a portion or region, preferably a single contiguous region, within a single larger polypeptide.

Preferred fragments include, for example, truncated polypeptides having a portion of the amino acid sequence of SEQ ID NO: 2 or variants thereof, for example comprising a contiguous series of residues of the amino-terminal and or carboxy-terminal amino acid sequence. Degraded forms of the polypeptides of the invention produced with or in a host cell are also preferred. More preferred are fragments with structural or functional features, e.g. containing alpha helices and alpha helix forming regions, beta sheets and beta sheet forming regions, turns and turn forming regions, coils and coil forming regions, hydrophilic regions, hydrophobic regions , alpha amphipathic region, beta amphipathic region, flexible region, surface forming region, substrate binding region and fragments of highly antigenic region.

More preferred fragments include isolated polypeptides comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 consecutive amino acids of the amino acid sequence of SEQ ID NO: 2; or an isolated polypeptide comprising the amino acid sequence , the amino acid sequence has at least 15, 20, 30, 40, 50 or 100 consecutive amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2.

Fragments of polypeptides of the invention are useful in the preparation of corresponding full-length polypeptides by peptide synthesis; thus, these fragments are useful as intermediates in the preparation of full-length polypeptides of the invention.

Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted or added in any combination.

A polypeptide or immunogenic fragment of the invention may be in the form of a "mature" protein, or may be part of a larger protein such as a precursor or fusion protein. It is generally preferred to include secretory or leader sequences, pre-sequences, sequences to aid in purification such as polyhistidine residues, or additional sequences to stabilize during recombinant production. Furthermore, the addition of exogenous polypeptide or lipid tails or polynucleotide sequences to increase the immunogenicity of the final molecule is also contemplated.

In one aspect, the present invention relates to a soluble fusion protein prepared by genetic engineering, which contains a polypeptide of the present invention or a fragment thereof and the heavy chain or light chain of each subclass of immunoglobulin (IgG, IgM, IgA, IgE). Various parts of the constant region of the chain. Preferably, the immunoglobulin is part of the constant region of human IgG, especially IgG1, and the fusion occurs at the hinge region. In a particular embodiment, the Fc portion can be removed simply by introducing a cleavage sequence that is cleavable with coagulation factor Xa.

Furthermore, the present invention relates to methods for preparing these fusion proteins by genetic engineering, and to their use in drug screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.

Proteins can be chemically conjugated, or expressed as recombinant fusion proteins that can be produced at higher levels in one expression system than non-fusion proteins. The fusion partner can help provide a T helper epitope (immune fusion partner), preferably a T helper epitope that can be recognized by humans; or a T helper epitope that can help the protein to be expressed at a higher yield than the original recombinant protein bit (expression enhancer). The fusion partner is preferably both an immunological fusion partner and an expression enhancer ligand.

Fusion partners include protein D from Haemophilus influenzae and the nonstructural protein NS1 (hemagglutinin) from influenza virus. Another fusion partner is a protein known as LytA. Preference is given to using the C-terminal part of the molecule. LytA comes from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase --- LytA amidase (encoded by the lytA gene {"Gene" 43 (1986) 265-272 pages}), the latter is An autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal region of the LytA protein is responsible for the affinity for choline or certain choline analogs such as DEAE. This property has been used to develop an E. coli C-LytA expression plasmid that can be used for expression of fusion proteins. The purification of a hybrid protein containing a C-LytA fragment at its amino terminus has been described {"Biotechnology" Vol. 10 (1992) pp. 795-798}. The repeat portion found at the C-terminus of the LytA molecule, starting from residue 178, eg residues 188-305, can be used.

The invention also includes variants of the aforementioned polypeptides, ie polypeptides which differ from a reference by conservative amino acid substitutions, wherein one residue is replaced by another residue with similar characteristics. Typically such substitutions are between the following amino acids: Ala, Val, Leu, and Ile; Ser and Thr; acidic residues Asp and Glu; Asn and Gln; basic residues Lys and Arg; or aromatic residues Phe and Tyr .

Polypeptides of the invention may be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Methods for preparing such polypeptides are well known in the art.

More preferably the polypeptide of the invention is from Neisseria meningitidis, but it is also preferably obtainable from other organisms of the same taxonomic genus. Polypeptides of the invention may also be obtained, for example, from the same taxonomic class or organism of interest. polynucleotide

It is an object of the present invention to provide polynucleotides encoding a BASB055 polypeptide, in particular a polynucleotide encoding a polypeptide referred to herein as BASB055.

In a particularly preferred embodiment of the present invention, the polynucleotide comprises a region encoding a BASB055 polypeptide, which region comprises a sequence listed in SEQ ID NO: 1, which includes a full-length gene or variants thereof.

The BASB055 polynucleotide provided in SEQ ID NO: 1 is the BASB055 polynucleotide from Neisseria meningitidis strain ATCC13090.

A further aspect of the present invention provides isolated nucleic acid molecules encoding and/or expressing BASB055 polypeptides and polynucleotides, particularly BASB055 polypeptides and polynucleotides of Neisseria meningitidis, including such as unprocessed RNAs, ribozyme RNAs , mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments of the present invention include polynucleotides and polypeptides useful biologically, diagnostically, prophylactically, clinically or therapeutically and compositions containing them.

Another aspect of the invention relates to isolated polynucleotides comprising at least one full-length gene encoding a BASB055 having a deduced amino acid sequence of SEQ ID NO: 2, and polynucleotides closely related thereto and variants thereof.

In another particularly preferred embodiment of the present invention, there is provided a BASB055 polypeptide from Neisseria meningitidis comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or a variant thereof.

Using the information provided here, such as the polynucleotide sequence set forth in SEQ ID NO: 1, polynucleotides encoding BASB055 polypeptides of the present invention can be obtained using standard cloning and screening methods, such as those used for cloning and screening from bacteria. The method for sequencing chromosomal DNA fragments uses Neisseria meningitidis as a starting material and subsequently obtains a full-length clone. For example, to obtain a polynucleotide sequence of the invention, such as the polynucleotide sequence given in SEQ ID NO: 1, a radiolabeled oligonucleotide, preferably a 17-mer or Longer, chromosomal DNA clone libraries of N. meningitidis in E. coli or other suitable hosts are screened. Clones carrying the same DNA as the probe are then differentiated using stringent hybridization conditions. By sequencing the individual clones thus identified by hybridization with sequencing primers designed on the basis of the original polypeptide or polynucleotide sequence, it is possible to extend the polynucleotide sequence in both directions, thereby determining the full-length gene sequence. Such sequencing is routinely performed using denatured double-stranded DNA, such as prepared from plasmid cloning. Suitable techniques are found in Maniatis, T., Fritsch, E.F. and Sambrook et al., "A Laboratory Guide to Molecular Cloning, Second Edition," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). (For details, see hybridization screening 1.90 and denatured double-stranded DNA template sequencing 13.70). Direct genomic DNA sequencing can also be used to obtain full-length gene sequences. To illustrate the present invention, each polynucleotide listed in SEQ ID NO: 1 was found from a DNA library from Neisseria meningitidis.

Furthermore, each DNA sequence listed in SEQ ID NO: 1 contains an open reading frame encoding a protein containing the number of amino acid residues listed in SEQ ID NO: 2, the deduced molecular weight of which can be derived using this The molecular weight values of amino acid residues well known to those skilled in the art were used for calculation.

The polynucleotide of SEQ ID NO: 1 is located between the nucleotide number 1 of the start codon of SEQ ID NO: 1 and the stop codon starting from nucleotide number 1237, and encodes the polypeptide of SEQ ID NO: 2.

In a further aspect, the present invention provides an isolated polynucleotide, the nucleotide comprising or consisting of: (a) a polynucleotide sequence, which is separated from SEQ ID NO: 1 in SEQ ID NO: 1 is at least 85% identical, more preferably at least 90% identical, more preferably at least 95% identical, most preferably at least 97-99% identical or completely identical over the entire length of 1; or (b) a A polynucleotide sequence encoding a polypeptide having at least 85% identity, more preferably at least 90% identity to the amino acid sequence of SEQ ID NO: 2 respectively over the entire length of SEQ ID NO: 2, More preferably at least 95% identity, more preferably at least 97-99% identity or 100% complete identity.

A polynucleotide encoding a polypeptide of the invention, including homologues or homologous evolutions from species other than Neisseria meningitidis, can be obtained by a method comprising: under stringent hybridization conditions Suitable libraries are screened (e.g. using a temperature range of 45-65° C. at a concentration of 0.1-1% SDS) with a labeled or detectable probe comprising or composed of SEQ ID NO: 1 sequence or a fragment thereof, and isolating a full-length gene and/or genomic clone containing said polynucleotide sequence.

The present invention provides a polynucleotide sequence identical to the coding sequence (open reading frame) of SEQ ID NO: 1 over its entire length. The present invention also provides the coding sequence of the mature polypeptide or fragment thereof alone and within a reading frame of another coding sequence, such as encoding a leader or secretory sequence, The sequence of a pre-, pro- or prepro-protein. The polynucleotides of the invention may also contain at least one non-coding sequence, including but not limited to, such as at least one non-coding 5' and 3' sequence, e.g. transcribed but not translated sequences, termination signals (e.g. rho-dependent and rho-independent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals. A polynucleotide sequence may also comprise additional coding sequences encoding additional amino acids. For example, a tag sequence that facilitates purification of the fusion polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a 6-histidine peptide provided by the pQE vector (Qiagen, Inc.), see Gentz et al., PNAS 86:821-824, (1989) or an HA peptide tail (Wilson et al., "Cell" 37:767 (1984); both tag sequences can be used to purify polypeptides fused to them. Polynucleotides of the present invention also include, but are not limited to , comprising a structural gene and its naturally associated sequences controlling gene expression.

The nucleotide sequence encoding the BASB055 polypeptide of SEQ ID NO: 2 may be identical to the polypeptide encoding sequence contained in nucleotides 1 to 1236 of SEQ ID NO: 1. Alternatively, it may be a sequence that also encodes the polypeptide of SEQ ID NO: 2 due to the redundancy (degeneracy) of the genetic code.

As used herein, the term "polynucleotide encoding a polypeptide" relates to a polynucleotide comprising a sequence encoding a polypeptide of the invention, in particular a bacterial polypeptide, more specifically meningeal Neisseria BASB055 polypeptide, which has the amino acid sequence listed in SEQ ID NO:2. The term also relates to a polynucleotide comprising a single contiguous or non-contiguous region encoding the peptide (e.g. integrated phage, integrated insert sequence, integrated vector sequence, integrated transposon sequence or Polynucleotides disrupted by RNA editing or genomic DNA rearrangement) and additional regions, which may also contain coding and/or non-coding sequences.

The present invention further relates to variants of the polynucleotides described herein which encode variants of the polypeptide having the deduced amino acid sequence of SEQ ID NO:2. Fragments of polynucleotides of the invention can be used, for example, to synthesize full-length polynucleotides of the invention.

A more particularly preferred embodiment is a polynucleotide encoding a BASB055 variant having several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acids in SEQ ID NO: 2 The amino acid sequence of the BASB055 polypeptide substituted, modified, deleted and/or added in any combination. Especially preferred among them are silent substitutions, additions and deletions which do not alter the properties and activities of the BASB055 polypeptide.

A more preferred embodiment of the present invention is a polynucleotide that is at least 85% identical to the polynucleotide encoding the BASB055 polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 over its entire length; acid-complementary polynucleotides. In this regard, polynucleotides having at least 90% identity over their entire length are particularly preferred, and among these particularly preferred polynucleotides, polynucleotides having at least 95% identity are more preferred, and Of these polynucleotides having at least 95% identity, at least 97% identical polynucleotides are more preferred, likewise at least 98% and at least 99% identical polynucleotides are especially more preferred, Polynucleotides that are at least 99% identical are more preferred.

A preferred embodiment is a polynucleotide encoding a polypeptide that substantially retains the same biological function or activity of the mature polypeptide encoded by the DNA of SEQ ID NO:1.

According to certain preferred embodiments of the present invention, the present invention provides polynucleotides capable of hybridizing to the BASB055 polynucleotide sequence such as the polynucleotide sequence of SEQ ID NO: 1, especially under stringent conditions.

The invention further relates to polynucleotide sequences that hybridize to the polynucleotide sequences provided herein. In this regard, the present invention particularly relates to polynucleotides capable of hybridizing under stringent conditions to the polynucleotides described herein. As used herein, "stringent conditions" and "stringent hybridization conditions" mean that hybridization occurs only when there is at least 95% and preferably at least 97% identity between the sequences. A specific example of stringent hybridization conditions is incubation overnight at 42°C in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured sheared salmon sperm DNA, followed by washing of the hybridization support in 0.1x SSC at approximately 65°C. Hybridization and washing conditions are well known and are exemplified in Sambrook et al., "A Laboratory Guide to Molecular Cloning, Second Edition," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989), especially Chapter 11. Solution hybridization can also be performed using the polynucleotide sequences provided by the present invention.

The present invention also provides polynucleotides comprising or consisting of a polynucleotide sequence obtained by screening a suitable library with a probe under stringent hybridization conditions and isolating said polynucleotide sequence wherein the library contains the complete gene of the polynucleotide sequence listed in SEQ ID NO: 1, and the probe has the sequence of said polynucleotide sequence listed in SEQ ID NO: 1. Fragments that can be used to obtain such a polynucleotide include probes and primers such as those detailed elsewhere herein.

As discussed elsewhere herein for polynucleotide assays, for example, polynucleotides of the invention can be used as hybridization probes for RNA, cDNA, and genomic DNA to isolate full-length cDNAs and genomic clones encoding BASB055, and Isolation of cDNA and genomic clones of other genes with high identity, especially high sequence identity, to the BASB055 gene. Such probes generally have at least 15 nucleotide residues or base pairs, such probes preferably have at least 30 nucleotide residues or base pairs, and may also have at least 50 nucleotide residues or base pairs. base pair. Particularly preferred probes have at least 20 nucleotide residues or base pairs and less than at least 30 nucleotide residues or base pairs.

The coding region of the BASB055 gene can be isolated by screening using the DNA sequence provided by SEQ ID NO: 1 to synthesize an oligonucleotide probe. A cDNA, genomic DNA or mRNA library is then screened using labeled oligonucleotides complementary to the gene sequences of the invention to determine the library members to which the probe hybridizes.

Several methods are available and well known to those skilled in the art for obtaining full-length DNAs or extending short DNAs, e.g. methods based on the Rapid Amplification of cDNA Ends (RACE) method (see e.g. Frohman et al., Proceedings of the National Academy of Sciences 85: 8998-9002, 1988). Recent modifications of this technique, such as the Marathon ^™ technology (Clontech Laboratories Inc.), significantly simplify the search for longer cDNAs. In Marathon ^(TM) technology, cDNAs are prepared from mRNA extracted from a selected tissue and a "linker" is attached to each end. The 5' end of the "missing" DNA is then amplified by nucleic acid amplification (PCR) using a combination of gene-specific and adapter-specific oligonucleotides. The PCR reaction is then repeated using "nested" primers, ie, primers designed to anneal to the interior of the amplified product (usually an adapter-specific primer that anneals to the 3' of the adapter sequence and to the gene sequence of choice). A gene-specific primer that anneals to the 5' of the The products of this reaction are then analyzed by DNA sequencing to generate a complete sequence by ligating the product directly to existing DNA, or by performing a separate full-length PCR using the new sequence information for designing 5' primers to construct a complete sequence. long DNA.

The polynucleotides and polypeptides of the present invention are useful as research reagents and substances such as in the discovery of disease, particularly in the treatment and diagnosis of human disease, as discussed herein in relation to polynucleotide assays.

The polynucleotides of the present invention are oligonucleotides derived from the sequences of SEQ ID NO: 1-2, which can be used in the methods presented here, but more preferably in PCR, to determine whether the polynucleotides identified here are all or Some can be transcribed in bacteria or infected tissue. It will be appreciated that such sequences can also be used to diagnose the stage of infection and the type of infection with the acquired pathogen.

The invention also provides polynucleotides encoding a polypeptide that is a mature protein plus additional amino- or carboxy-terminal amino acids, or amino acids within the mature polypeptide (e.g., when the mature form contains more than one polypeptide chain hour). Such sequences may function in the processing of a protein from a precursor to a mature form, may allow protein transport, may increase or decrease the half-life of the protein, or may facilitate manipulation of the protein for testing or manufacturing. Usually in vivo, additional amino acids are processed from the mature protein by cellular enzymes.

For each and all polynucleotides of the invention there is provided a complementary polynucleotide. These complementary polynucleotides are preferably fully complementary to each polynucleotide to which they are complementary.

A precursor protein comprising the mature form of a polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. This inactive precursor is generally activated when the prosequence is removed. Some or all of the prosequence may be removed prior to activation. This precursor is generally referred to as a proprotein.

In addition to the standard A, G, C, T/U representation of nucleotides, "N" can also be used to describe a specific polynucleotide of the present invention. "N" means that any of the 4 DNA or RNA nucleotides may occur at that specified position in the DNA or RNA sequence, but preferably N is not a nucleic acid when combined with adjacent nucleotide positions , when read in the correct reading frame, produces a pre-mature stop codon in that reading frame.

In summary, the polynucleotides of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a proprotein containing one or more leader sequences that are not preproteins, A precursor to a mature protein of a sequence, or a precursor to a proprotein, is a preproprotein having a leader sequence and one or more prosequences that are normally removed during processing to produce the active and mature forms of the polypeptide.

According to one aspect of the present invention there is provided the use of a polynucleotide of the present invention for therapeutic or prophylactic purposes, in particular genetic immunization.

The use of polynucleotides of the present invention in genetic immunization preferably uses a suitable delivery method, such as direct injection of plasmid DNA into muscle (Wolff et al. "Human Molecular Genetics" (1992) 1:363, Manthorpe et al., Human "Gene Therapy" (1983) 4: 419); delivery after complexing DNA with a specific protein carrier (Wu et al., "Journal of Biochemistry" (1989) 264: 16985), co-precipitating DNA with calcium phosphate (Benvenisty & Reshef, " Proceedings of the National Academy of Sciences (1986) 83:9551); encapsulation of DNA with various forms of liposomes (Kaneda et al., "Science" (1989) 243:375); particle bombardment (Tang et al., "Nature" (1992 ) 356:152, Eisenbraun et al. DNA Cell Biology (1993) 12:791) and in vivo infection using cloned retrovectors (Seeger et al. Proc. Vectors, host cells, expression systems

The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered with the vectors of the present invention, and polypeptides of the present invention produced by recombinant techniques. Cell-free translation systems can also be used to produce such proteins using RNAs from the DNA constructs of the invention.

The recombinant polypeptides of the present invention can be produced from genetically engineered host cells containing expression systems using methods well known to those skilled in the art. Accordingly, in a further aspect, the present invention relates to expression systems comprising polynucleotides of the invention, host cells genetically engineered with such expression systems, and recombinant techniques for producing polypeptides of the invention.

For recombinant production of the polypeptides of the present invention, host cells may be genetically engineered to incorporate expression systems or parts thereof or polynucleotides of the present invention. Introduction of polynucleotides into host cells can be accomplished by a variety of methods described in standard laboratory manuals, such as Davis et al., Fundamental Methods in Molecular Biology (1986) and Sambrook et al., A Laboratory Guide to Molecular Cloning, Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYork (1989), e.g. calcium phosphate transfection, DEAE-dextran mediated transfection, vector transvection, microinjection, cationic lipid mediated transfection, electroporation Perforation, transduction, scratch inoculation, bombardment introduction, and infection.

Representative examples of suitable hosts include bacterial cells such as Streptococcus, Staphylococcus, Enterococcus, Escherichia coli, Streptomyces, Cyanobacteria, Bacillus subtilis, Moraxella catarrhalis, Haemophilus influenzae and Neisseria meningitidis cells; fungal cells such as Kluyveromyces, Saccharomyces and other yeasts, Candida albicans and Aspergillus and other basidiomycete cells; insect cells Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa , C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells such as gymnosperm or angiosperm cells.

A variety of expression systems can be used to prepare the polypeptides of the invention. Such vectors include chromosomal, episomal, viral-derived vectors, e.g. derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papovaviruses such as SV40 , vaccinia virus, adenovirus, fowl pox virus, pseudorabies virus, picornavirus, retrovirus, and alphavirus-derived vectors or vectors derived from combinations thereof, such as vectors derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Expression system constructs may contain control regions that regulate as well as cause expression. In this regard generally, any system or vector suitable for maintaining, propagating or expressing a polynucleotide and/or expressing a polypeptide in a host can be used for expression. Suitable DNA sequences can be inserted into the expression system by any of the well known and routine techniques, such as those outlined in Sambrook et al., A Laboratory Guide to Molecular Cloning (supra).

In the recombinant expression system of eukaryotic cells, in order to secrete the translated protein into the endoplasmic reticulum, the periplasm or the extracellular environment, appropriate secretion signals can be integrated into the expressed polypeptide. These signals may be endogenous to the polypeptide, or may be heterologous.

Polypeptides of the invention can be recovered and purified from recombinant cell culture by well known techniques including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography , affinity chromatography, hydroxyapatite chromatography and lectin chromatography. More preferably metal ion affinity chromatography (IMAC) is used for purification. Well known techniques for protein refolding can be used to regenerate the active conformation when the polypeptide is denatured during intracellular synthesis, isolation and/or purification.

The expression system can also be a recombinant living microorganism, such as a virus or bacterium. The gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Vaccination and in vivo infection with this live vector will result in in vivo expression of the antigen and induction of an immune response. Viruses and bacteria used for this purpose are such as poxviruses (e.g. vaccinia virus, fowl pox virus, canary fowl pox virus), alphaviruses (Sindbis virus, Semliki Forest virus, Venezuelan equine encephalomyelitis virus) , adenovirus, adeno-associated virus, picornavirus (poliovirus, rhinovirus), herpesvirus (varicella zoster virus, etc.), Listeria, Salmonella, Shigella, Neisseria, BCG . These viruses and bacteria can be virulent, or attenuated in various ways to obtain a live vaccine. Such live vaccines are also part of the present invention. Diagnostic, prognostic, serotyping and mutation testing

The invention also relates to the use of the BASB055 polynucleotides and polypeptides of the invention as diagnostic reagents. Detection of BASB055 polynucleotides and/or polypeptides in eukaryotic cells, especially mammalian cells, especially human cells can provide a diagnostic method for diagnosing diseases, disease stages or the response of infected organisms to drugs. Eukaryotic cells, especially mammalian cells, especially human cells, especially those infected or suspected of being infected with organisms containing the BASB055 gene or protein, can be tested at the nucleic acid or amino acid level by a variety of well-known techniques and provided herein. method to detect.

Polypeptides and polynucleotides for prognostic, diagnostic or other analysis may be obtained from presumed infections and/or body material of infected individuals. Polynucleotides, especially DNA or RNA, from any of these sources can be used directly for detection, or can be amplified enzymatically using PCR or any other amplification technique prior to analysis. RNA, especially mRNA, cDNA and genomic DNA can also be used in the same manner. Using amplification methods, the species and strains of infectious or resident organisms present in an individual can be identified by genotyping selected polynucleotides of the organism. Deletions and insertions can be detected by size changes of the amplified products compared to a genotype selected from a reference sequence of a related organism, preferably a different species of the same genus or a different strain of the same species. Point mutations can be identified by hybridizing amplified DNA to labeled BASB055 polynucleotide sequences. For DNA or RNA, very or significantly matched sequences can be distinguished from poorly matched or significantly mismatched duplexes by DNase or RNase digestion, respectively, or by detecting differences in melting temperature or renaturation kinetics . Polynucleotide sequence differences can also be detected by changes in the electrophoretic mobility of polynucleotide fragments in a gel compared to a reference sequence. This can be done with or without denaturing reagents. Polynucleotide differences can also be detected by direct DNA or RNA sequencing. See, eg, Myers et al., Science 230:1242 (1985). Sequence changes at specific positions can also be revealed by nuclease protection assays such as RNase, V1 and S1 protection assays or by chemical cleavage methods. See, eg, Cotton et al. Proceedings of the National Academy of Sciences 85: 4397-4401 (1985).

In another embodiment, a series of oligonucleotide probes containing the nucleotide sequence of BASB055 or its fragments can be constructed for effective screening such as genetic mutation, serotype, classification or identification. Parallel technology approaches are well known and general, and can be used to address a variety of problems in molecular genetics, including gene expression, genetic linkage, and genetic variation (see, eg, Chee et al. Science 274:610 (1996).

Therefore, in another aspect, the present invention relates to a diagnostic kit comprising

(a) a polynucleotide of the present invention, preferably a nucleotide sequence of SEQ ID NO: 1 or a fragment thereof;

(b) a nucleotide sequence complementary to the nucleotide sequence of (a);

(c) a polypeptide of the present invention, preferably a polypeptide of SEQ ID NO: 2 or a fragment thereof; or

(d) antibodies against the polypeptide of the present invention, preferably against the polypeptide of SEQ ID NO: 2.

In any such kit, (a), (b), (c) or (d) may preferably contain an essential component. Such kits can be used in the diagnosis or otherwise of a disease or susceptibility to a disease.

The invention also relates to the use of the polynucleotides of the invention as diagnostic reagents. Detection of a mutant form of a polynucleotide of the invention, preferably SEQ ID NO: 1, associated with a disease or pathogenicity provides a diagnostic means that increases or limits the Diagnosis of a disease, prediction of disease process, disease stage, or determination of disease susceptibility resulting from expression, overexpression, or expression alteration. Organisms, particularly infectious organisms, carrying mutations in such polynucleotides can be detected at the polynucleotide level by various techniques such as those described anywhere herein.

Cells from organisms with mutations or polymorphisms (allelic mutations) in the polynucleotides and/or polypeptides of the invention can also be used to perform detection at the polynucleotide or polypeptide level using a variety of techniques, such as Serological typing. For example, RT-PCR can be used to detect mutations in RNA. Preferably RT-PCR is used in conjunction with an automated detection system such as GeneScan. RNA, cDNA or genomic DNA can also be used for the same purpose - PCR. For example, PCR primers complementary to a polynucleotide encoding a BASB055 polypeptide can be used to identify and analyze mutations.

The invention further provides primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or 3' ends. These primers can be used with other materials to amplify BASB055 DNA and/or RNA isolated from a sample obtained from an individual, such as body material. These primers can be used to amplify a polynucleotide isolated from an infected individual so that the polynucleotide can subsequently be elucidated by various techniques to elucidate the polynucleotide sequence. By this method, mutations in polynucleotide sequences can be detected and used to diagnose and/or predict infection or its stage or course, or to serotype and/or classify infectious agents.

The present invention further provides a method of diagnosing a disease, preferably a bacterial infection, more preferably an infection caused by Neisseria meningitidis, which comprises detecting a virus having the sequence of SEQ ID NO: 1 in a sample obtained from an individual, such as a body material. Increased expression levels of polynucleotides. An increase or decrease in the expression of a BASB055 polynucleotide can be detected using any method known in the art for polynucleotide quantification, such as amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrophotometry, and other hybridization methods .

Furthermore, a diagnostic assay according to the invention for detecting overexpression of a BASB055 polypeptide compared to a normal control tissue sample can be used to detect, for example, the presence of an infection. Assay techniques that can be used to determine the level of BASB055 polypeptide in a material from a host, such as a body, are well known to those skilled in the art. Such assay methods include radioimmunoassays, competitive binding assays, Western blots, antibody sandwich assays, antibody detection, and ELISA assays.

The polynucleotides of the invention can be used as components of polynucleotide arrays, preferably high density arrays or grids. These high-density arrays are particularly useful for diagnostic and prognostic purposes. For example, a set of spots each containing a different gene and further containing a polynucleotide of the invention can be used for probe detection, e.g., using hybridization or nucleic acid amplification, using probes from or derived from a body sample. The presence in an individual of a particular oligonucleotide sequence or related sequences. This presence can indicate the presence of a pathogen, particularly N. meningitidis, and can be used to diagnose and/or predict a disease or disease process. Preferred is a grid comprising variants of the nucleotide sequence of SEQ ID NO: 1. Also preferred are grids comprising multiple variants of the polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 2. Antibody

Polypeptides and polynucleotides of the present invention or variants thereof, or cells expressing them, can be used as immunogens to raise antibodies against such polypeptides or polynucleotides, respectively.

In certain preferred embodiments of the invention, antibodies directed against BASB055 polypeptides or polynucleotides are provided.

Antibodies prepared against the polypeptides or polynucleotides of the present invention can be obtained by using conventional methods, that is, the polypeptides and/or polynucleotides of the present invention, or an epitope-bearing fragment of one or both of them, one of them or An analog of both, or a cell expressing either or both of them, is administered to an animal, preferably a non-human animal. Any technique in the art that provides antibodies produced in continuous cell line culture can be used. Examples include techniques such as Kohler, G. and Milstein, C. Nature 256:495-497 (1975); Kozbor et al Immunology Today 4:72 (1983); Cole et al Monoclonal Antibodies and Cancer Therapy", pp. 77-96 of Alan R. Liss, Inc. (1985).

Techniques for making single-chain antibodies (US Patent No. 4,946,778) can be adapted to make single-chain antibodies to polypeptides or polynucleotides of the invention. Transgenic mice or other organisms or animals such as other mammals can also be used to express humanized antibodies immunospecific for the polypeptides or polynucleotides of the present invention.

In addition, phage display technology can be used to select antibody genes (McCafferty et al. (1990) "Nature" 348: 552-554; Marks et al. (1992) Biotechnology 10:779-783). The affinity of these antibodies can also be increased by techniques such as strand displacement (Clackson et al. (1991) Nature 352:628).

The antibodies described above can be used to isolate or identify clones capable of expressing the polypeptides or polynucleotides of the present invention, to purify these polypeptides or polynucleotides by, for example, affinity chromatography.

These antibodies, as well as other antibodies directed against BASB055 polypeptides or BASB055 polynucleotides, can be used to treat infections, particularly bacterial infections.

Polypeptide variants include antigenic, epitopic or immunologically equivalent variants and form a particular aspect of the invention.

Antibodies or fragments thereof are preferably modified to be less immunogenic in an individual. For example, if the individual is human, the antibody may more preferably be a "humanized" antibody, in which the complementarity determining regions of a hybridoma-derived antibody are grafted into a human monoclonal antibody, e.g. Jones et al. (1986) Nature 321 , 522-525 or the introduction in Tempest et al. (1991) Biotechnology 9, 266-273. Antagonists and Agonists --- Assays and Molecules

The polypeptides and polynucleotides of the invention can also be used to evaluate the binding of small molecule substrates and ligands in, for example, cells, cell-free extracts, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands, or may be structural or functional mimics, see eg Coligan et al. Current Methods in Immunology 1(2):Chapter 5 (1991).

Screening methods can simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes carrying the polypeptide or polynucleotide, or to fusion proteins of the polypeptide, via a label directly or indirectly linked to the candidate compound. In addition, screening methods may include competition with a labeled competitor. Furthermore, these screening methods can detect whether a candidate compound results in a signal resulting from activation of the polypeptide or polynucleotide, using a detection system appropriate to the cells containing the polypeptide or polynucleotide. Inhibitors of activation are generally tested in the presence of a known agonist and the effect of the candidate compound on the activation of the agonist is observed. Constitutively active polypeptides and/or constitutively expressed polypeptides and polynucleotides can be used, as desired, in the absence of an agonist or antagonist, to reverse the screening method for agonists or inhibitors, which can be detected by detecting candidate It is determined whether the compound causes inhibition of the activation of the polypeptide or polynucleotide. Moreover, the screening method may simply include the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention to form a mixture; detecting the activity of the BASB055 polypeptide and/or polynucleotide in the mixture ; comparing the activity of BASB055 polypeptides and/or polynucleotides in the mixture with a standard. Fusion proteins such as those prepared from the Fc portion of the present invention and the BASB055 polypeptide can also be used in high-throughput screening assays to identify antagonists of the polypeptides of the present invention and phylogenetically and/or functionally related polypeptides (see D. Bennett et al. J. Molecular Recognition 8:52-58 (1995) and K. Johanson et al. J. Biochem. 270(16):9459-9471 (1995)).

Polynucleotides, polypeptides and antibodies capable of binding and/or interacting with a polypeptide of the invention may also be used in the design of screening methods to detect the effect of the added compound on mRNA and/or polypeptide production in cells. For example, an ELISA assay can be designed using monoclonal and polyclonal antibodies to measure the level of secretion or cellular association of the polypeptide according to methods standard in the art. This can be used to discover agents that inhibit or enhance polypeptide production in appropriately manipulated cells or tissues (also known as antagonists or agonists, respectively).

The present invention also provides a method of screening compounds for identifying compounds capable of enhancing (agonist) or blocking (antagonist) the effect of BASB055 polypeptide or polynucleotide, especially compounds capable of inhibiting and/or killing bacteria. This screening method can use high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mixture, a cellular component such as a membrane, cell envelope, or cell wall, or any preparation thereof, comprising a BASB055 polypeptide and a labeled substrate or such The ligand for the polypeptide is incubated in the presence or absence of a candidate molecule that may be an agonist or antagonist of BASB055. The ability of a candidate compound to agonize or antagonize a BASB055 polypeptide is reflected by reduced binding of the labeled ligand or reduced production of a product of such a substrate. Molecules that bind ineffectively, ie do not induce the effects of the BASB055 polypeptide, are likely to be good antagonists. An agonist is a molecule that binds well and increases the rate at which a product is produced from a substrate, increases signal transduction, or increases the activity of a chemical channel, as the case may be. Depending on the circumstances, detection of the rate or level of product generation from a substrate, signaling, or chemical channel activity can be enhanced through the use of a reporter system. Reporter systems that can be used for this purpose include, but are not limited to, colorimetric, labeled substrate to product conversion, a reporter gene responsive to changes in the BASB055 polynucleotide or polypeptide, and binding assays well known in the art.

Another example of a BASB055 agonist assay is a competition assay that combines BASB055 and a potential agonist with a BASB055-binding molecule, a recombinant BASB055-binding molecule, a natural substrate or ligand, or a mimic of a substrate or ligand Combination of compounds, under appropriate conditions for competition inhibition test. BASB055 can be labeled, eg, with a radioactive or colorimetric compound, so that the number of BASB055 molecules bound to a binding molecule or converted to a product can be accurately measured to evaluate the effect of a potential antagonist.

Potential antagonists include small organic molecules, peptides, polypeptides, and antibodies, among others, that are capable of binding to the polynucleotides and/or polypeptides of the invention and thereby inhibiting or suppressing their activity or expression. Potential antagonists may also be small organic molecules, peptides, polypeptides such as closely related proteins or antibodies that bind at the same site of the binding molecule, e.g. a binding molecule that does not induce the activity induced by BASB055, thereby passing Preventing the binding of the BASB055 polypeptide and/or polynucleotide inhibits the activation or expression of the BASB055 polypeptide and/or polynucleotide.

Potential antagonists include small molecules that bind and occupy the binding site of the polypeptide, thereby preventing its binding to the cellular binding molecule and thereby inhibiting normal biological activity. Examples of small molecules include, but are not limited to, small organic molecules, peptides or peptide-like molecules. Other possible antagonists include antisense molecules (see Okano, Journal of Neurochemistry 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988) , an introduction to these molecules). Preferred potential antagonists include compounds related to BASB055 and variants thereof.

In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention or fragments thereof and heavy chains or Various portions of the light chain constant region. A preferred immunoglobulin is the constant region portion of a human IgG, particularly IgGl heavy chain, wherein fusion occurs at the hinge region. In a particular embodiment, the Fc portion can be removed simply by introducing a cleavage sequence which allows blood clotting factor Xa. Furthermore, the present invention relates to methods for preparing these fusion proteins by genetic engineering, and to their use in drug screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.

Each polynucleotide sequence provided here can be used in the discovery and development of antimicrobial compounds. The encoded protein can be used as a target for screening antibacterial drugs through expression. In addition, polynucleotide sequences encoding the amino-terminal region of the encoded protein or the SD or other translation-promoting region of the corresponding mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.

The invention also provides the use of a polypeptide, polynucleotide, agonist or antagonist of the invention for interfering with the interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host susceptible to secondary symptoms of infection. Physiological interactions initially. In particular, the molecules of the invention are useful for: Inhibiting the adhesion of bacteria, in particular Gram-positive and/or Gram-negative bacteria, to extracellular matrix proteins or adhesion of eukaryotic, preferably mammalian, deeply buried apparatus extracellular matrix proteins in wounds; blocking bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial BASB055 proteins, which mediate tissue damage; and/or blocking whether deeply buried Device implantation is also a normal pathogenic process for infections caused by other surgical techniques.

According to another aspect of the present invention, there are provided BASB055 agonists and antagonists, preferably bacteriostatic or bactericidal agonists and antagonists.

Antagonists and agonists of the invention are useful, for example, in the prevention, suppression and/or treatment of diseases.

In a further aspect, the invention relates to mimotopes of the polypeptides of the invention. A mimotope is a peptide sequence that is sufficiently similar (in sequence or structure) to a native peptide that it is recognized by an antibody that recognizes the native peptide, or is capable of raising antibodies that recognize the native peptide when coupled to a suitable carrier.

Peptide mimotopes can be used for a particular purpose by adding, deleting or substituting selected amino acids. Thus, peptides can be modified to facilitate attachment to a protein carrier. For example, for some chemical conjugation methods, it is desirable to contain a terminal cysteine. In addition, for peptide binding to a protein carrier, it is desirable to include a hydrophobic terminus away from the peptide-binding terminus, so that the unconjugated free terminus of the peptide remains in interaction with the surface of the carrier protein. The peptide is thus given a conformation that closely resembles the conformation the peptide has throughout its natural molecular structure. For example, a peptide can be modified to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail. Additionally, addition or substitution of the D stereoisomer of one or more amino acids may be performed to prepare a useful derivative, eg, to enhance the stability of the peptide.

Furthermore, peptide mimotopes can be identified by techniques such as phage display (EP 0 552 267 B1 ) using antibodies capable of binding the polypeptides of the invention. This technique produces a large number of peptide sequences that mimic the structure of the native peptide and are thus capable of binding antibodies against the native peptide, but may not have significant sequence homology to the native peptide. vaccine

Another aspect of the present invention relates to a method of inducing an immune response in an individual, particularly a mammal, preferably a human, which method comprises vaccinating the individual with a BASB055 polynucleotide and/or polypeptide or fragment or variant thereof sufficient to produce antibodies and and/or a T cell immune response to protect the individual from infection, particularly bacterial infection, especially Neisseria meningitidis infection. The invention also provides methods of generating such an immune response to delay bacterial replication. Another aspect of the present invention relates to a method of inducing an immune response in an individual comprising administering to such an individual a nucleic acid vector, sequence or ribozyme that directs the expression of a BASB055 polynucleotide and/or polypeptide, or a fragment or variant thereof, to expressing BASB055 polynucleotides and/or polypeptides or fragments or variants thereof in vivo, thereby inducing an immune response, e.g., production of antibodies and/or T cell immune responses, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from a disease, whether the disease is already present in the individual or not. An example of gene delivery is as a coating on particles or other substances to facilitate its entry into desired cells. Such nucleic acid vectors may include DNA, RNA, ribozymes, modified nucleic acids, DNA/RNA hybrids, DNA-protein complexes, or RNA-protein complexes.

A further aspect of the present invention relates to an immune composition which, when introduced into an individual, preferably a human, is capable of inducing an immune response in the individual against and/or encoded by the BASB055 polynucleotide. Peptide immune response. Wherein the composition comprises recombinant BASB055 polynucleotides and/or polypeptides encoded thereby; and/or DNA comprising antigens capable of encoding and expressing said BASB055 polynucleotides, polypeptides encoded thereby or other polypeptides of the present invention and and/or RNA. The immune response can be used for therapeutic or prophylactic purposes, and can take the form of antibody immunity and/or cellular immunity, eg, by CTL or CD4+ T cells.

Thus, a BASB055 polypeptide or fragment thereof may be fused to an accessory protein or chemical moiety, which may or may not be capable of producing antibodies itself, but which stabilizes the first protein and produces a fused or modified protein, The latter are antigenic and/or immunogenic, preferably protective. Such fusion recombinant protein preferably further contains an antigenic accessory protein, such as lipoprotein D from Haemophilus influenzae, glutathione-S-transferase (GST) or β-galactosidase or any other capable of stabilizing Proteins and large accessory proteins that facilitate their preparation and purification. Furthermore, the accessory protein can be used as an adjuvant in providing a standard stimulus to the immune system of the organism receiving the protein. Accessory proteins can be attached to the amino- or carboxyl-terminus of the first protein.

In the vaccine composition of the present invention, the BASB055 polypeptide and/or polynucleotide or fragments thereof, mimotopes, or variants may be present in a carrier, such as a live recombinant carrier such as the above-mentioned live bacterial carrier.

Also suitable are inanimate vectors of the BASB055 polypeptide, such as bacterial outer membrane vesicles or "blebs". OM vesicles are derived from the outer membrane of the double membrane of Gram-negative bacteria and have been reported in a variety of Gram-negative bacteria including C. trachomatis and C. psittaci (Zhou, L et al., 1998, FEMS Microbiology Letters, 163: 223-228). Non-limiting examples of bacterial pathogens reported to produce vesicles also include: Bordetella pertussis, Borrelia brucei, Brucella malta, Brucella ovis, Escherichia coli, Haemophilus influenzae, Legionella pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, and Yersinia enterocolitica.

Vesicles have the advantage of presenting outer membrane proteins in their native conformation and are therefore particularly useful in vaccines. Vesicles can also be used in vaccines by engineering bacteria to alter the expression of one or more molecules on the outer membrane. Thus, for example, outer membrane expression of desired immunogenic proteins, such as the BASB055 polypeptide, can be introduced or upregulated (eg, by altering the promoter). Alternatively or further, the inclusion of outer membrane molecules that are irrelevant (such as non-protective antigens or immunodominant but variable proteins) or deleterious (such as toxic molecules such as LPS, or potential inducers of autoimmune responses) can be down. These methods are described in detail below.

The non-coding flanking region of the BASB055 gene contains important regulatory elements in gene expression. This regulation exists at both the transcriptional and translational levels. The sequences of these regions (whether upstream or downstream of the gene's open reading frame) can be obtained by DNA sequencing. This sequence information allows identification of potential regulatory motifs such as distinct promoter elements, terminator sequences, inducible sequence elements, repressors, elements responsible for phase transition, ribosome binding sequences, potential secondary structures with involvement in regulation , and other types of regulatory motifs or sequences. This sequence is another aspect of the invention.

This sequence information allows regulation of native expression of the BASB055 gene. Upregulation of gene expression may also be achieved by altering promoters, ribosome binding sequences, potential repressor or operator elements or any other relevant elements. Likewise, downregulation of expression can be achieved by similar regulation. Alternatively, by altering the phase transition sequence, the expression of a gene can be placed under phase transition control, or can be uncoupled from such regulation. Alternatively, the expression of the gene can be placed under the control of one or more inducible elements that allow regulation of expression. Examples of such modulation include, but are not limited to, induction by temperature shifts, addition of induction substrates such as selected carbohydrates or derivatives thereof, trace elements, vitamins, cofactors, metal ions, and the like.

The above changes can be imported in several different ways. Altering sequences involved in gene expression can be achieved by in vivo random mutagenesis followed by selection for the desired phenotype. Another approach is to isolate the region of interest and alter it by random or site-directed substitution, insertion or deletion mutagenesis. The altered region can be reintroduced into the bacterial genome by homologous recombination, and the effect on gene expression can be evaluated. Alternatively, sequence knowledge of a region of interest can be used to replace or delete all or part of a native regulatory sequence. In such cases, the regulatory region of interest is isolated and altered to include regulatory elements from other genes, a combination of regulatory elements from different genes, a synthetic regulatory region, or any other regulatory region, or to delete selected portions of the wild-type regulatory sequence . These altered sequences can be reintroduced into the bacterial genome by homologous recombination. Non-limiting examples of preferred promoters that can be used to upregulate gene expression include the promoters proA, proB, lbpB, tbpB, p110, lst, hpuAB from Neisseria meningitidis or Neisseria gonorrhoeae; from M. Catarrhalis TbpB from Haemophilus influenzae; p1, p2, p4, p5, p6, lpD, tbpB, D15, Hia, Hmw1, Hmw2 from Haemophilus influenzae.

In one example, gene expression can be altered by exchanging its promoter for a stronger promoter (by isolating the upstream sequence of the gene, modifying the sequence in vitro, and reintroducing it into the genome by homologous recombination). Upregulated expression can be achieved in bacteria as well as outer membrane vesicles from the bacteria.

In one example, the above approach can be used to generate recombinant bacterial strains with improved performance in vaccine applications. These can be attenuated strains, strains with increased expression of selected antigens, strains with gene knockouts (or reduced expression) that interfere with the immune response, strains with altered expression of immunodominant proteins, strains with altered shedding of outer membrane vesicles, But not limited to this.

Accordingly, the present invention also provides altered upstream regions of the BASB055 gene, these altered regions also containing heterologous regulatory elements that alter the expression level of the BASB055 protein located on the outer membrane protein. The upstream region of this aspect of the invention includes the upstream sequence of the BASB055 gene. The upstream region begins upstream of the BASB055 gene and generally extends to less than about 1000 bp upstream of the ATG start codon. When the gene is in a polycistronic sequence (operon), the upstream region can start just before the gene of interest, or before the first gene in the operon. Preferably, the altered upstream region of this aspect of the invention contains a heterologous promoter located 500 to 700 bp upstream of the ATG.

Accordingly, the present invention provides BASB055 polypeptides in altered bacterial vesicles. The invention also provides modified host cells capable of producing non-living membrane-based vesicle vectors. The present invention also provides a nucleic acid vector containing a BASB055 gene with an altered upstream region containing a heterologous regulatory element.

The invention also provides methods of making the host cells and bacterial vesicles of the invention.

The present invention also provides compositions, particularly vaccine compositions, comprising the polypeptides and/or polynucleotides of the present invention and immunomodulatory DNA sequences such as those described in Sato, Y. Science 273:352 (1996), and method.

The present invention also provides methods for using the described polynucleotides or specific fragments thereof in polynucleotide constructs used in such genetic immunization experiments in animal models of Neisseria meningitidis infection, wherein the polynucleotides or Particular fragments have been shown to encode non-variable regions of bacterial cell surface proteins. Such experiments are particularly useful for identifying protein epitopes capable of eliciting a prophylactic or therapeutic immune response. It is believed that this method will enable the subsequent preparation of monoclonal antibodies of particular value from the necessary organs of animals that have successfully resisted or cleared the infection for the development of mammals, especially humans, for the treatment of bacterial infections, especially A prophylactic or therapeutic agent for Neisserial infection.

The invention also includes vaccine formulations comprising an immunogenic recombinant polypeptide and/or polynucleotide of the invention and a suitable carrier, such as a pharmaceutically acceptable carrier. Since polypeptides and polynucleotides may be disrupted in the stomach, they are preferably administered parenterally, including, for example, subcutaneously, intramuscularly, intravenously, or intradermally. Formulations suitable for parenteral administration include aqueous and non-aqueous non-aqueous Bacterial injection solutions, which may contain antioxidants, buffers, bacteriostatic agents and solvents capable of rendering the preparation isotonic with the body fluids of the individual, preferably blood, and aqueous and non-aqueous sterile suspensions which may contain suspending or thickening agents . The preparations can be placed in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried environment, requiring only the addition of a sterile liquid carrier just before use.

The vaccine formulations of the present invention may also include adjuvant systems to enhance the immunogenicity of the formulations. Preferably the adjuvant system preferentially elicits a TH1 type response.

Immune responses can be roughly divided into two typical types, namely humoral or cell-mediated immune responses (generally distinguished by their protective antibodies and cellular effector mechanisms, respectively). These types of responses are referred to as TH1-type responses (cell-mediated responses) and TH2-type immune responses (humoral responses).

A typical TH1-type immune response is characterized by the generation of antigen-specific haplotype-restricted cytotoxic T lymphocytes and natural killer cell responses. In mice, TH1-type responses are often characterized by the production of antibodies of the IgG2a subtype, whereas in humans their counterparts are IgG1-type antibodies. A TH2-type immune response is characterized by the production of a broad spectrum of immunoglobulin isotypes, including IgG1, IgA, and IgM in mice.

Conceivably, the driving forces behind these two types of immune responses are cytokines. High levels of TH1-type cytokines preferentially induce a cell-mediated immune response against a given antigen, while high levels of TH2-type cytokines preferentially induce a humoral immune response against an antigen.

The distinction between TH1 and TH2 type immune responses is not absolute. In fact, an individual can support an immune response described as TH1-dominant or TH2-dominant. However, it is often convenient to consider the family of cytokines as described by Mosmann and Coffman in the murine CD4+ve T cell clone (Mosmann, T.R. and Coffman, R.L. (1989), TH1 and TH2 cells: Different secretion patterns of lymphokines lead to Different functional properties. Annals of Immunology vol. 7, pp. 145-173). Typically, TH1-type responses are associated with production of INF-γ and IL-2 cytokines by T lymphocytes. Other cytokines such as IL-12, which are usually directly related to the induction of TH1-type immune responses, are not produced by T cells. In contrast, TH2-type responses are associated with the secretion of IL-4, IL-5, IL-6 and IL-13.

Certain vaccine adjuvants are known to be particularly suitable for stimulating TH1 or TH2 type cytokine responses. The best indicators of the TH1:TH2 balance of the immune response following vaccination or infection usually include direct measurement of TH1 or TH2 cytokine production by T lymphocytes after restimulation with antigen in vitro, and/or IgG1: IgG2a ratio.

Thus, TH1-type adjuvants preferentially stimulate isolated T cell populations to produce high levels of TH1-type cytokines and promote CD8+ cytotoxic T lymphocytes and antigen-specific immunity associated with TH1-type isotypes upon restimulation with antigen in vitro Adjuvant produced by globulin response.

Adjuvants capable of preferentially stimulating TH1 cell responses are described in International Patent Application Nos. WO94/00153 and WO95/17209.

3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is known from GB2220211 (Ribi). It is chemically a mixture of 3 De-O acylated monophosphoryl lipid A with 4, 5 or 6 acyl chains and is manufactured by Ribi Immunochem, Montana. 3 A preferred form of De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 (SmithKline Beecham Biologicals SA).

The 3D-MPL particles are preferably small enough to be sterilized by filtration through a 0.22 μm microporous membrane (EP Patent 0 689 454).

3D-MPL is present in the range of 10 μg-100 μg per dose, preferably 20-25 μg, while antigen is usually present in the range of 2-50 μg per dose.

Another preferred adjuvant comprises QS21, an Hplc purified non-toxic fraction from the stem of Quillaja Saponaria Molina. It is optionally available in combination with 3 De-O-acylated monophosphoryl lipid A (3D-MPL) and can be used with a carrier.

A method of preparing QS21 is disclosed in US Patent No. 5,057,540.

Non-reactive adjuvant formulations containing QS21 have been described previously (WO96/33739). This formulation containing QS21 and cholesterol has been shown to be a successful TH1 stimulating adjuvant when formulated with antigen.

Further adjuvants which preferentially stimulate TH1 -type cellular responses include immunomodulatory oligonucleotides such as unmethylated CpG sequences as disclosed in WO96/02555.

Combinations of different TH1 stimulatory adjuvants such as those described above are also considered to provide an adjuvant that preferentially stimulates a TH1 type cellular response. For example, QS21 can be formulated in combination with 3D-MPL. The ratio of OS21:3D-MPL is usually 1:10 to 10:1, preferably 1:5 to 5:1, and usually about 1:1. The preferred optimal combination range is 2.5:1 to 1:1 3D-MPL:QS21.

According to the invention, the vaccine composition also preferably contains a carrier. The carrier can be an oil-in-water emulsion, or an aluminum salt such as aluminum phosphate or hydroxide.

Oil-in-water emulsions preferably contain a metabolizable oil such as squalene, alpha-tocopherol and Tween 80. In a particularly preferred aspect, according to the invention, the antigens in the vaccine composition are combined with QS21 and 3D-MPL in such an emulsion. Additionally, the oil-in-water emulsion may contain span 85 and/or lecithin and/or tricaprylin.

When administered to humans, the range of QS21 and 3D-MPL in the vaccine is 1 μg-200 μg per dose, such as 10-100 μg, preferably 10 μg-50 μg. Oil-in-water typically contains 2 to 10% squalene, 2 to 10% alpha-tocopherol, and 0.3 to 3% Tween 80. The ratio of squalene:alpha-tocopherol:Tween 80 is preferably the same or less than 1 when provided in a more stable emulsion. Span85 may also be included at a level of 1%. In some cases, the vaccine of the present invention preferably further contains a stabilizer.

Non-toxic oil-in-water emulsions preferably contain a non-toxic oil such as squalane or squalene, an emulsifier such as Tween 80 in an aqueous carrier. The aqueous carrier can be, for example, phosphate buffered saline.

A particularly potent adjuvant formulation containing QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO95/17210.

The invention also provides a multivalent vaccine composition comprising the vaccine formulation of the invention together with other antigens, particularly antigens useful in the treatment of cancer, autoimmune diseases and related conditions. Such a multivalent vaccine composition may include a TH1 inducible adjuvant as previously described.

Although the present invention describes specific BASB055 polypeptides and polynucleotides, it should be understood that they cover naturally occurring polypeptides and polynucleotides as well as similar polypeptides and polynucleotides containing additions, deletions or substitutions, which additions, deletions or Substitutions do not substantially affect the immunogenic characteristics of the recombinant polypeptide or polynucleotide.

Antigens can also be delivered as whole bacteria (dead or alive) or as subcellular components, these possibilities include N. meningitidis itself. Compositions, kits and administration

In a further aspect of the invention there is provided a composition comprising a BASB055 polynucleotide and/or a BASB055 polypeptide for administration to a cell or a multicellular organism.

The present invention also relates to compositions comprising the polynucleotides and/or polypeptides discussed herein, or agonists or antagonists thereof. The polypeptides and polynucleotides of the invention may be used in combination with a non-sterile or sterile carrier or carrier for a cell, tissue or organism, such as a pharmaceutical carrier suitable for administration to an individual. Such compositions include, for example, a vehicle additive or a therapeutically effective dose of a polypeptide and/or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the above-mentioned components of the invention.

Polypeptides, polynucleotides and other compounds of the invention may be used alone or in combination with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered by any effective conventional means, including such as by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes, and other routes.

For therapeutic or prophylactic use, the active agent may be administered to a subject in the form of an injectable composition, eg, as a sterile aqueous dispersion, preferably in isotonic form.

In a further aspect, the present invention provides pharmaceutical compositions comprising a therapeutically effective dose of a polypeptide and/or polynucleotide such as a soluble form, agonist or antagonist of a polypeptide and/or polynucleotide of the invention Or a small molecular compound, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the above-mentioned ingredients of the invention. Polypeptides, polynucleotides and other compounds of the invention may be used alone or in combination with other compounds, such as therapeutic compounds.

The composition should be administered by one route, for example by a systemic or oral route. A preferred form of systemic administration involves injection, usually intravenously. Other routes of injection such as subcutaneous, intramuscular or peritoneal can be used. Other methods of systemic administration include transmucosal and dermal administration using penetrants such as bile salts or fusidic acid or other detergents. Furthermore, if a polypeptide or other compound of the invention can be formulated as an enteric or a capsule formulation, the oral route can also be used. Administration of these compounds can also be transdermal and/or topically, in the form of ointments, patches, gels, solutions, powders and the like.

For administration to mammals, especially humans, the daily dosage level of the active agent should be from 0.01 mg/kg to 10 mg/kg, usually around 1 mg/kg. In any case, the physician should determine the actual dosage which will be most suitable for the individual and will vary according to the age, weight and response of a particular individual. In individual cases, of course, higher or lower dosage ranges may be employed and such are within the scope of this invention.

The desired dosage range will depend on the peptide chosen, the route of administration, the nature of the formulation, the nature of the user's condition and the judgment of the physician. However, suitable dosages are in the range of 0.1-100 [mu]g per kg.

Vaccine compositions are generally in injectable form. Traditional adjuvants can be used to enhance the immune response. A suitable unit dose for vaccination is 0.5-5 μg/kg of antigen, and such doses are preferably administered 1-3 times at intervals of 1-3 weeks. For the indicated dosage range, no toxic side effects are observed when the compounds of the invention are administered to suitable individuals.

The required dosage will vary widely, however, taking into account that different compounds may be used and that different routes of administration may produce different effects. For example, the oral route requires higher doses than intravenous injection. Variations in these dosage levels can be adjusted according to methods well known in the art using standard approaches to optimization practice. Sequence databases, sequences in tangible media, and algorithms

Polynucleotide and polypeptide sequences constitute a valuable source of information for the determination of their two- and three-dimensional structures and for the further identification of similarly homologous sequences. These methods are conveniently performed by storing them on a computer readable medium and then storing the data into a known macromolecular structure program or searching a sequence database using well known search tools such as the GCG package.

The present invention also provides methods for analyzing characteristic sequences or chains, especially gene sequences or coding protein sequences. Preferred methods for sequence analysis include: methods such as sequence homology analysis such as identity and similarity analysis, DNA, RNA and protein structure analysis, sequence alignment, evolution analysis, sequence motif analysis, open reading frame determination, nucleic acid base Calling, codon usage analysis, nucleic acid base sorting, and sequence chromatography peak analysis.

The present invention provides a computer-based method for homology identification. The method comprises the steps of: providing a first polynucleotide sequence comprising a polynucleotide sequence of the invention in a computer readable medium, combining said first polynucleotide with at least one second Compare polynucleotide or polypeptide sequences to determine homology.

The present invention provides a computer-based method for homology identification. The method comprises the steps of: providing a first polypeptide sequence comprising a polypeptide sequence of the invention in a computer readable medium, combining said first polypeptide sequence with at least one second polynucleotide Or polypeptide sequence comparison to determine homology.

All publications and references, including but not limited to patents and patent applications, cited in this application are hereby incorporated by reference in their entirety as if each were specifically and individually indicated to be set forth herein in its entirety. Any patent application from which this application claims priority is also incorporated herein by reference in its entirety in the same manner as publications and references described above. definition

"Identity", as is well known in the art, is the relatedness between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by sequence comparison. In the art, "identity" also refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the matching of strands of these sequences. "Identity" can be conveniently calculated using known methods, including but not limited to ("Computational Molecular Biology", edited by Lesk, A.M., Oxford University Press, New York, 1988; "Biological Computers: Information and Genome Project" Smith , D.W., ed., Academic Press, New York, 1993; "Computer Analysis of Sequence Data," Part I, edited by Griffin, A.M. and Griffin, H.G., HumanaPress, New Jersey, 1994; "Sequence Analysis in Molecular Biology," von Heine, G., Academic Press, 1987; and "Primers for Sequence Analysis", edited by Gribskov, M. and Devereux, J., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., "SIAM J. Applied Math "48: 1073(1988). The method for determining identity is designed to give the maximum match between the detected sequences. And, the method for determining identity is coded in a publicly available computer program. Used to determine two sequences Computer program methods for identity between include, but are not limited to, the GAP program (Devereux, J. et al. Nucleic Acids Research 12(1): 387 (1984), BLASTP, BLASTN (Altschul, S.F. et al. Biology Journal 215:403-410 (1990) and FASTA (Pearson and Lipman Proceedings of the National Academy of Sciences 85:2444-2448 (1988). The BLAST family of programs is available from NCBI and other sources (The BLAST Handbook, Altschul, S. et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S. et al. J. Molecular Biology 215: 403-410 (1990). The well-known Smith Waterman algorithm can also be used to determine identity.

Parameters for polypeptide sequence comparisons include the following:

Algorithms: Needleman and Wunsch, Journal of Molecular Biology 48:443-453 (1970)

Comparison matrix: Henikoff and Henikoff's BLOSSUM62

Proceedings of the National Academy of Sciences 89: 10915-10919 (1992)

Notch Compensation: 8

Notch Length Compensation: 2

A program using these parameters is available from the Genetics Computer Group, Madison WI, as a "gap" program. The above parameters are the default parameters for polypeptide comparisons (no compensation for end gaps).

Parameters for comparison of polynucleotide sequences include the following:

Algorithms: Needleman and Wunsch, Journal of Molecular Biology 48:443-453 (1970)

Comparison Matrix: Match = +10, No Match = 0

Notch Compensation: 50

Notch Length Compensation: 3

Source: Genetics Computer Group, Madison WI's "Gap" program. The above parameters are the default parameters for nucleic acid comparisons.

Preferred meanings of "identity" for polynucleotides and polypeptides are provided in (1) and (2) below, as appropriate.

(1) The polynucleotide embodiment further includes an isolated polynucleotide comprising a nucleotide sequence having at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1, or may include a certain number of nucleotide changes compared to the reference sequence, wherein such changes may be selected from at least one nucleotide deletion, substitution (including transitions and transversions) or insertion, wherein said alteration may occur at the 5' or 3' end of the reference polynucleotide sequence or at any position between the two ends, Interspersed between nucleotides of the reference sequence, respectively, or in one or more contigs in the reference sequence, wherein the number of nucleotide changes is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by The corresponding percent identity (divided by 100) is then subtracted from the total nucleotide number of SEQ ID NO: 1, or:

n _n ≤ x _n -(x _n y)

where n _n is the number of nucleotide changes, x _n is the total number of nucleotides in SEQ ID NO: 1, y is 0.50 for 50%, 0.60 for 60%, and 0.70 for 70% , is 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97%, 1.00 for 100%, and ·It is the symbol of multiplication operation. If the result of x _n and y is not an integer, it will be rounded to the nearest integer and then subtracted from x _n . Changes in the polynucleotide encoding the polypeptide of SEQ ID NO: 2 may produce nonsense, missense or frameshift mutations in the coding sequence, so that the polypeptide encoded by the altered polynucleotide will change.

For example, the polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 1, that is, the identity is 100%, or it may include a certain number of nucleotide changes compared with the reference sequence, so the identity is less than 100%. Such alterations may be selected from at least one nucleotide deletion, substitution (including transitions and transversions) or insertion, wherein said alteration may occur at the 5' or 3' end or between the two ends of the reference polynucleotide sequence Any position, respectively interspersed between nucleotides of the reference sequence, or interspersed in one or more contigs in the reference sequence. The number of nucleotide changes is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the corresponding percent identity (divided by 100), and then dividing the result from the total number of nucleotides in SEQ ID NO: 1 Subtract, or:

n _n ≤ x _n -(x _n y)

Where n _n is the number of nucleotide changes, x _n is the total nucleotide number of SEQ ID NO: 1, and the y value is 0.70 at 70%, 0.80 at 80%, 0.85 at 85%, and so on By analogy, · is the sign of the multiplication operation, if the result of x _n and y is not an integer, it will be rounded to the nearest integer, and then subtracted from x _n .

(2) The polypeptide embodiment further includes an isolated polypeptide comprising a polypeptide sequence that is at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identical to the reference sequence of SEQ ID NO: 2 wherein the polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2, or may include a certain number of amino acid changes compared to the reference sequence, wherein the changes are selected from at least one amino acid deletion, substitution (including conservative or non-conservative substitutions) or insertions, and said alterations may occur at or anywhere in between the amino- or carboxy-terminal positions of the reference polypeptide sequence, interspersed respectively among the amino acids of the reference sequence, or in one or more contigs Interspersed in the reference sequence, and the number of said amino acid changes is determined as follows: the total number of amino acids in SEQ ID NO: 2 is multiplied by the corresponding percent identity (divided by 100), and the result is then calculated from the total number of amino acids in SEQ ID NO: 2 Subtract from the amino acid count, or:

n _a ≤ x _a -(x _a y)

Wherein n _a is the number of amino acid changes, x _a is the total number of amino acids in SEQ ID NO: 2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, and 0.70 for 80%. say 0.80, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97%, 1.00 for 100%, and multiplication sign, if the result of x _a and y is not an integer, it is rounded to the nearest integer and subtracted from x _a .

For example, the polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 2, that is, the identity is 100%, or it may include a certain number of amino acid changes compared with the reference sequence, so the identity is less than 100%. Such alterations may be selected from at least one amino acid deletion, substitution (including conservative and non-conservative substitutions) or insertion, wherein said alteration may occur at any position at the amino or carboxyl terminus of the reference polypeptide sequence or between the two termini, interspersed respectively Between amino acids of the reference sequence, or interspersed in the reference sequence in one or more contigs. The number of amino acid changes for a given percent identity is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the corresponding percent identity (divided by 100), and then subtracting the result from the total number of amino acids in SEQ ID NO: 2. except, or:

n _a ≤ x _a -(x _a y)

Wherein n _a is the number of amino acid changes, x _a is the total amino acid number of SEQ ID NO: 2, the y value is 0.70 at 70%, 0.80 at 80%, 0.85 at 85%, and so on, while · is the symbol for multiplication, where if the result of x _a and y is not an integer, it is rounded to the nearest integer and subtracted from x _a .

"Individual" when used herein to refer to an organism refers to a multicellular eukaryote including, but not limited to, metazoans, mammals, oviparous animals, bovids, apes, primates, and humans.

"Isolated" means altered "by the hand of man" from its natural state, ie, if an "isolated" composition or substance occurs in nature, it has been altered or removed from its original environment or has been removed and altered. For example, a polynucleotide or polypeptide present in a living body is not "isolated" when the term is used herein, but the same polynucleotide or polypeptide that has been separated from coexisting substances in a natural state is "isolated". Furthermore, a polynucleotide or polypeptide is "isolated" when it is introduced into an organism, by transformation, genetic manipulation, or any other recombinant method, even if it is still present in said organism, which may be live or die.

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be modified or unmodified RNA or DNA, including single- and double-stranded regions.

"Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. Typically, a polynucleotide variant differs in nucleotide sequence from a reference polynucleotide. Changes in the nucleotide sequence of a variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. As described below, nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Typically, a polypeptide variant differs in amino acid sequence from a reference polypeptide. Typically, the differences are limited such that the sequences of the reference polypeptide and the variant are generally very similar and identical in many regions. A variant and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be an amino acid encoded by the genetic code. Variants of a polynucleotide or polypeptide may be naturally occurring, such as allelic variants, or non-naturally occurring. Non-naturally occurring polynucleotide and polypeptide variants can be prepared by mutagenesis techniques or direct synthesis.

"Disease" means any disease caused by or associated with a bacterial infection, including, for example, upper respiratory tract infections, invasive bacterial diseases such as bacteremia and meningitis.

Example

The following examples were performed using standard techniques, which are well known and routine to those skilled in the art unless they are described in detail elsewhere. The examples are illustrative, not restrictive of the invention. Example 1: BASB055 sequence in N. meningitidis serotype B strain ATCC13090

The sequence of the BASB055 gene of Neisseria meningitidis strain ATCC13090 is shown in SEQ ID NO:1. The translation result of the BASB055 polynucleotide sequence shown in SEQ ID NO: 2 has amino acid sequence homology with the MtrC protein of Neisseria gonorrhoeae. The BASB055 polypeptide contains a signal sequence characteristic of lipoproteins. Example 2: Construction of a plasmid expressing recombinant BASB055 A: Cloning of BASB055

Insert NdeI and XhoI restriction sites into forward Lip11-Fm/p (5′-AGG CAGAGG CAT ATG GCT TTT TAT GCT TTT AAG GCG ATG CG-3′, SEQ ID NO: 3) and reverse Lip11-RCf/p respectively (5′-AGG CAG AGG CTC GAG TTC CGCTTC AGA AGC AGT TTT GGC TTC-3′, SEQ ID NO: 4) amplification primers, which allow the directional cloning of BASB055 PCR products into the low-copy Escherichia coli expression plasmid pTLZ2, making BASB055 The protein can be expressed as a fusion protein containing (His)6 affinity chromatography marker at the C-terminus. The BASB055 PCR product was purified from the amplification product using a silica-based spin column (QiaGen) according to the manufacturer's instructions. To generate the NdeI and XhoI ends required for cloning, the purified PCR product was sequentially digested to completion with NdeI and XhoI restriction enzymes according to the manufacturer's (Life Technologies) instructions.

After the first restriction digest, the PCR product was purified as above by spin column to remove salts and eluted with sterile water before the second enzymatic digestion. Digested DNA fragments were repurified using silica-based spin columns before ligation with the pTLZ2 plasmid. B: Expression vector preparation

To prepare the expression plasmid pTLZ2 for ligation, it was similarly digested to completion with NdeI and XhoI, and then treated with calf intestinal phosphatase (CIP, about 0.02 units/pimol 5' end, Life Technologies) according to the manufacturer's instructions to prevent self-ligation. An approximately 5-fold molar excess of the digested fragment relative to the prepared vector was used for the ligation reaction. A standard 20 μl ligation reaction (approximately 16°C, approximately 16 hours) was performed using T4 DNA ligase (approximately 2.0 units/reaction, Life Technologies) using techniques known in the art. An aliquot of the ligation reaction (approximately 5 μl) was used to transform electrocompetent BL21 DE3 cells according to techniques known in the art. After growing in about 1.0 ml of LB medium at 37°C for about 2-3 hours, the transformed cells were plated on LB agar plates containing ampicillin (100 μg/ml). Antibiotics were included in the selection medium to ensure that all transformed cells harbored the pTLZ2 plasmid (ApR). Plates were incubated at 37°C for approximately 16 hours. Individual ApR colonies were picked out with a sterile toothpick, and "flaked" were inoculated into fresh LB ApR plates and about 1.0ml LB ApRR culture medium. The flake plate and the culture solution were incubated at 37°C in a standard incubator (plate) or on a water bath shaker.

Whole-cell PCR analysis was performed to confirm that transformants contained the BASB055 DNA insert. About 1.0 ml of LB Ap overnight culture was transferred to a 1.5 ml polypropylene tube and the cells were collected by centrifugation in a Beckman centrifuge (about 3 minutes, room temperature, about 12000 x g). Cell pellets were resuspended in approximately 200 μl sterile water and approximately 10 μl aliquots were used to perform PCR reactions with a final volume of approximately 50 μl containing BASB055 forward and reverse amplification primers. The final concentrations of the PCR reaction components were essentially the same as in Example 2, but about 5 units of Taq polymerase were used. The initial 95°C denaturation step was increased to 3 minutes to ensure thermal lysis of bacterial cells and release of plasmid DNA. Use an ABI 9700 thermal cycler to perform 32 cycles of 3-step thermal cycling. The cycling conditions are: 95°C for 45 seconds; 55-58°C for 45 seconds; 72°C for 1 minute to amplify the BASB055 PCR fragment of the lysed transformant sample . After thermal cycling, approximately 20 [mu]l reaction aliquots were taken for analysis by agarose gel electrophoresis (0.8% agarose in Tris-acetic acid-EDTA (TAE) buffer). DNA fragments were visualized after gel electrophoresis using UV irradiation and ethidium bromide staining. DNA molecular weight standards (1 Kb gradient, Life Technologies) were electrophoresed in parallel with the samples to estimate the size of PCR products. Transformants producing expected PCR products were identified as strains containing the BASB055 expression construct. The induced expression of recombinant BASB055 in strains containing the expression plasmid was then analyzed. C: Expression analysis of PCR positive transformants

For each PCR-positive transformant identified above, about 5 ml of LB medium containing ampicillin (100 μg/ml) was inoculated with cells from the sheet plate and grown overnight at 37° C. with shaking (about 250 rpm). An aliquot (approximately 1.0 ml) of the overnight seed culture was inoculated into a 125 ml flask containing approximately 25 ml of LB Ap broth and grown overnight at 37°C with shaking (approximately 250 rpm) until the culture turbidity reached an O.D.600 of approximately 0.5 , the mid-log phase (usually about 1.5 to 2.0 hours). At this point, approximately normal culture (approximately 12.5 ml) was transferred to a second 125 ml flask, and expression of recombinant BASB055 protein was induced by adding IPTG (1.0 M stock solution prepared in sterile water, Sigma) to a final concentration of 1.0 mM. IPTG-induced and non-induced cultures were incubated with shaking at 37°C for an additional approximately 4 hours. Samples (approximately 1.0 ml) of induced and uninduced cultures were removed after the induction period and cells were harvested by centrifugation in a centrifuge for approximately 3 minutes at room temperature. Individual cell pellets were suspended in approximately 50 μl sterile water, then mixed with an equal volume of 2×Laemmli SDS-PAGE sample buffer containing 2-mercaptoethanol, and placed in a boiling water bath for approximately 3 minutes to denature proteins. Equal volumes (about 15 μl) of crude lysates of IPTG-induced and uninduced cells were loaded on two 12% Tris/glycine polyacrylamide gels (1 mm thick Mini-gels, Novex). Induced and uninduced lysate samples were electrophoresed with pre-stained molecular weight markers (SeeBlue, Novex) under conventional conditions using standard SDS/Tris/Glycine running buffer (BioRad). After electrophoresis, one gel was stained with Coomassie brilliant blue R250 (BioRad) and then destained to reveal the new BASB055 IPTG-induced protein. The second gel was transferred to a PVDF membrane (0.45 micron pore size, Novex) by electroblotting at 4°C for approximately 2 hours using a BioRad Mini-Protean II blotting apparatus and Towbin methanol (20%) transfer buffer. Membrane blocking and antibody incubation are performed according to techniques known in the art. A monoclonal anti-(His)5 antibody was used followed by a rabbit anti-mouse antibody conjugated to HRP (QiaGen) to confirm the expression and identity of the BASB055 recombinant protein. Anti-His antibody reactivity was characterized using ABT insoluble substrate or Hyperfilm using the Amersham ECL chemiluminescence system. Embodiment 3: Recombinant BASB055 lipidation (lipidation) state

To determine whether the BASB055 recombinant protein was lipidated, radiolabeling experiments were performed using ³ H-glycerol and ³ H-palmitic acid as lipid-specific incorporation markers. Cultures of BL21DE3 E. coli strains containing expression plasmids were grown on M9 minimal medium and pulse-labeled with 50 μCi of ³ H-glycerol or ³ H-palmitic acid during a 3-hour IPTG induction. After washing to remove unincorporated isotope, whole cell lysates of radiolabeled cultures were run on gradient polyacrylamide gels (4-20%), fixed, and dried. Autoradiograms were generated by exposing the xerogels to Hyperfilm (Amersham) at 70°C for about 7 days. Both markers produced visible bands of the same size as IPTG-induced Coomassie blue-stainable proteins. ^{The 3} H-label incorporated into the IPTG-induced protein supports the conclusion that the BASB055 protein is somewhat lipidated in E. coli. Example 4: Production of recombinant BASB055

bacterial strain

Recombinant expression in E. coli BL21 DE3 harboring the pTLZ2 plasmid encoding N. meningitidis BASB055 was used to generate a cell population of purified recombinant protein. Expression strains were cultured on LB agar plates containing 100 μg/ml ampicillin (Ap) to ensure the presence of the plasmid. For storage at -80°C, strains were propagated in LB broth containing the same concentration of antibiotics, and then mixed with an equal volume of LB broth containing 30% (w/v) glycerol.

culture medium

The fermentation medium used to produce recombinant proteins was 2X YT broth (Difco) containing 100 μg/ml Ap. Antifoam was added to the medium in the fermenter to 0.25ml/L (Antifoam204, Sigma). To induce the expression of BASB055 recombinant protein, IPTG (Isopropyl β-D-thiogalactopyranoside) (1 mM, final concentration) was added to the fermentor.

fermentation

A 500 ml seed culture flask containing a 50 ml working volume was inoculated with 0.3 ml of a rapidly thawed frozen culture or a few colonies from a selective agar plate on a shaker (Innova 2100, New Brunswick Scientific) at 150 rpm at 37+ Incubate at 1°C for about 12 hours. This seed culture was used to inoculate a fermenter with a working volume of 5 liters containing 2X YT broth and Ap. The operating conditions of the fermenter (Bioflo 3000, New Brunswick Scientific) were 37 ± 1 °C, 0.2-0.4 VVM aeration, Rushton impeller 250 rpm. The pH in flask seed cultures and fermentors did not need to be controlled. During fermentation, the pH in the fermenter was 6.5 to 7.3. When the culture reached the mid-log phase of growth (O.D.600 about 0.7 units), IPTG (1.0 M stock solution, prepared in sterile water) was added to the fermentor. Cells were induced for 2-4 hours and then harvested by centrifugation using a 28RS Heraeus (Sepatech) or RC5C ultracentrifuge (Srovall Instruments). Cell pellets were stored at -20°C until use.

purification

Imidazole and biotech grade or higher reagents were obtained from Ameresco Chemical, Solon, Ohio. Triton X-100 (tert-octylphenoxypolyethoxyethanol), Triton X-114, sodium monobasic phosphate, and urea were all reagent grade or better and were obtained from Sigma Chemical Company, St. Louis, Missouri. Dulbecco's phosphate buffered saline (1X PBS) was obtained from Quality Biological, Inc., Gaithersburg, Maryland. Dulbecco's phosphate buffered saline (10X PBS) was obtained from Bio Whittaker, Walkersville, Maryland. BSA-free Penta-His antibody was obtained from QiaGen, Valencia, California. Peroxidase-conjugated affinity pure grade goat anti-mouse IgG was obtained from Jackson Immuno Research, West Grove, Penn. All other reagents were reagent grade or better.

Ni chelating Sepharose fast flow resin was obtained from Pharmacia, Sweden. Precast Tris-Glycine 4-20% and 10-20% polyacrylamide gels, all running buffers and solutions, SeeBlue pre-stained standards, multi-labeled multi-color standards, and PVDF transfer membranes were obtained from Novex, San Diego, California. SDS-PAGE silver staining kit was obtained from Daiichi Pure Chemicals Company Limtid, Toyko, Japan. Coomassie staining solution was obtained from Bio-Rad Laboratories, Hercules, California. Acrodisc(R) PF 0.2m syringe filters were obtained from Pall Gelman Science, Ann Arbor, Michigan. GD/X 25 mm disposable syringe filters were obtained from Whatman Inc., Clifton, New Jersey. Dialysis tubing 8,000 MWCO was obtained from BioDesign Inc. Od New York, Carmak New York. BCA protein assay reagents and Snake Skin dialysis tubing 3,500 MWCO were obtained from Pierce Chemical Co. Rockford, Illinois.

extraction steps

Cell pellets were thawed at room temperature for 30 to 60 minutes. Weigh 5 to 6 grams of material into a 50 mL disposable centrifuge tube. Recombinant BASB055 antigen was purified by extraction of cell membranes with 1.0% Triton X114 and phase separation at 37°C based on the cloud point of Triton X114. The Triton X114 phase was diluted with 50 mM Tris-HCl containing 10% glycerol, 5% ethylene glycol and 0.5% Triton X100. It was loaded on a Ni-chelated Sepharose fast-flow column. The protein was then eluted with 200 mM imidazole to affinity-purify the histidine-tagged protein to obtain more than 90% pure protein.

final formulation

BASB055 was dialyzed overnight against 0.1% Triton X-100 and 1×PBS, pH 7.4, with 3 dialysate changes. Purified proteins were identified and used to generate antibodies as described below.

Biochemical identification: SDS-PAGE and Western blot analysis

Recombinant purified proteins were separated on 4-20% polyacrylamide gels and electrotransferred to PVDF membranes at 100 V for 1 hour as previously described (Thebaine et al., 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354). The PVDF membrane was then pretreated with 25 ml of Dulbecco's phosphate buffered saline containing 5% non-fat dry milk. All further incubations were performed using this pretreatment buffer.

PVDF membranes were incubated with anti-His tail antibody dilution for 1 hour at room temperature. Then wash the membrane twice with washing buffer (20mM Tris buffer, pH7.5, containing 150mM sodium chloride and 0.05% Tween-20). The PVDF membrane was then incubated with 25 ml of a 1:5000 dilution of peroxidase-labeled species-specific conjugate for 30 minutes at room temperature. The PVDF membrane was then washed 4 times with wash buffer and developed with 3-amino-9-ethylcarbazole and urea peroxide supplied by Zymed (San Francisco, CA).

SDS-PAGE results ( FIG. 1A ) showed a protein of about 50 kDa with a purity greater than 90%, which reacted with anti-tetra-His antibody in SDS-PAGE Western blot ( FIG. 1B ). Example 5: Immunization of mice with recombinant BASB055

Partially purified recombinant BASB055 protein expressed in E. coli was injected three times into BalB/C mice (10 animals/group) on days 0, 14 and 28. Animals were injected subcutaneously with approximately 5 μg of antigen per dose, which was prepared in two different formulations: either adsorbed on 100 μg aluminum phosphate, or formulated in SBAS2 emulsion (SB62 emulsion containing 5 μg MPL and 1 μg QS21 per dose) . A negative control group consisting of mice immunized with SBAS2 emulsion only was also added to the experiments. The mice were bled on day 28 (14 days PostII) and 35 days (7 days PostIII) to detect specific anti-BASB055 antibodies. The specific anti-BASB055 antibody was determined by Elisa of partially purified BASB055 protein and E. coli protein. Antibody responses were also performed by Western blot on different N. meningitidis B strains. Pooled sera (from 10 mice/group) from the formulation group (7 days PostIII only) were assayed in these experiments. The results shown in Figure 2 clearly show that the antibody response was excellent, whereas the anti-E. coli antibody response (Figure 3), although also positive, was considerably lower than the specific BASB055 response (Figure 2). Aluminum phosphate preparations induced the highest antibody levels. Western blots of immunized mouse sera shown in Figures 4 and 5 confirmed that BASB055 protein was clearly visible at the expected 50 kDa MW.

Recognition of BASB055 epitopes on different Neisseria meningitidis strains by Western blot

In this assay, sera from immunized mice (pooled sera) were detected by Western blot to recognize the BASB055 epitope on 7 different N. meningitidis B strains as well as on partially purified recombinant BASB055 proteins, 7 The strains are: H44/76 (B: 15: P1.7, lineage ET-5), M97 250987 (B: 4: P1.15), BZ10 (B: 2b: P1.2, common lineage A4), BZ198 ( B:NT ^* :-, lineage 3), EG328 (B:NT ^* , lineage ST-18), NGP165 (B:2a:P1.2, ET 37 cluster) and ATCC 13090 (B:15:P1.15) Neisseria meningitidis B strain. (*: NT: not typed).

Briefly, 10 μl (>10 ⁸ cells/lane) of each sample treated with sample buffer (95°C for 10 minutes) was placed on an SDS-PAGE gradient gel (Tris-glycine 4-20%, Novex, code n°EC60252). Electrophoresis was performed at 125 volts for 90 minutes (35 mA/gel). Proteins were then transferred to nitrocellulose membranes (0.45 μm, code n° 162-0114) using the Bio-rad transfer system (code n° 170-3930) at 100 volts for 1:30 hours. Filters were blocked with PBS-0.05% Tween 20 overnight at room temperature and then incubated with mouse serum containing anti-BASB055 antibody derived from aluminum phosphate and SBAS2 preparations. These sera were diluted 100-fold in PBS-0.05% Tween 20, and incubated on nitrocellulose membranes with shaking at room temperature for 2 hours. Wash the nitrocellulose membrane in PBS-0.05% Tween 20 for 5 minutes, repeat this step 3 times, and then wash the nitrocellulose membrane with the appropriate conjugate (biotinylated Goat anti-mouse Ig antibody, Amersham code n°RPN1001) was incubated for 1 hour at room temperature with gentle shaking. Membranes were washed 3 times as previously described and incubated with streptavidin-peroxidase complex (Amersham code n° 1051) diluted 1/1000 in washing buffer for 30 minutes with shaking. After the last 3 repeated washing steps, color development was performed by incubating for 20 min in a 50 ml solution containing 30 mg 4-chloro-1-naphthol (Sigma), 10 ml methanol, 40 ml PBS and 30 _{μl H2O2} . Color development was terminated by washing the membrane several times in distilled water.

The results in Figures 4 and 5 show that all tested strains reacted with a major band of BASB055 around 50 kDa, with a few bands of lower molecular weight, which are likely related to the antigen as they were absent in the E. coli preparation. These results suggest that the BASB055 protein may be expressed in all N. meningitidis serotype B strains. In Figure 4 and Figure 5, the recombinant BASB055 protein recognized by mouse serum has the same molecular weight. However, the smallest band (<9 kDa) recognized by mouse serum appeared to be associated with E. coli contamination, as evidenced by the last lane of the E. coli extract (Figure 5).

Sequence information BASB055 polynucleotide and polypeptide sequence SEQ ID NO: 1 BASB055 polynucleotide sequence ID NO of Neisseria meningitidis ATCC13090 strain ID NO: 2 Neisseria meningitidis BASB055 deduced from the polynucleotide of SEQ ID NO: 1多肽序列MAFYAFKAMRAALAAAVALVLSSCGKGGDAAQGGQPAGREAPAPVVGVVTVHPQTVALTVELPGRLESLRTADVRAQVGGIIQKRLFQEGSYVRAGQPLYQIDSSTYEANLESARAQLATAQATLAKADADLARYKPLVAAEAVSRQEYDAAVTAKRSAEAGVKAAQAAIKSAGINLNRSRITAPISGFIGQSKVSEGTLLNAGDTTVLATIRQTNPMYVNVTQSASEVMKLRRQIAEGKLLAADGVIAVGIKFDDGTVYPEKGRLLFADPVVNESTGQITLRAAVPNDQNILMPGLYVRVLMDQVAVDNAFVVPQQAVTRGAKDTVMIVNAQGGMEPREVTVAQQQGTNWIVTSGLKDGDKVVVEGISIAGITGAKKVTPKEWASSENQAAAPQSGVQTASEAKTASEAESEQ ID NO：3AGG CAG AGG CAT ATG GCT TTT TAT GCT TTT AAG GCG ATG CGSEQ ID NO：4AGG CAG AGG CTC GAG TTC CGC TTC AGA AGC AGT TIT GGC TTC

preservation material

A deposit containing the serotype B strain of Neisseria meningitidis was deposited with the American Type Culture Collection (referred to herein as "ATCC") on June 22, 1997 under accession number 13090. The deposit is Neisseria meningitidis (Albrecht and Ghon) and is a lyophilizate of a 1.5-2.9 kb insert library constructed from N. meningitidis isolates. This deposit is described in Int. Bull. Bacteriol. Nomencl. Taxon. 8:1-15 (1958).

This deposit of N. meningitidis strains is referred to herein as a "deposited strain" or "DNA of a deposited strain".

The deposited strain contains the full-length BASB055 gene. When the amino acid sequence of the polynucleotide contained in the deposited strain and any encoded polypeptide is inconsistent with the sequence description herein, the former shall prevail.

Deposited strains were deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. When the patent is granted, the strain can be released to the public without restrictions and conditions. The preserved strain is provided only for the convenience of those skilled in the art, and the practice of the present invention does not necessarily require the preserved material, as required by 35 U.S.C.ξ112.

Sequence Listing<110>SmithKline Beecham Biologicals S.A.<120>New Compound<130>BM45353<160>4<170>FastSEQ for Windows Version 3.0<210>1<211>1239<212>DNA<213>Nisserial meningitis菌(Neisseria meningitidis)<400> 1atggcttttt atgcttttaa ggcgatgcgt gcggccgcgt tggctgccgc cgttgcattg       60gtactgtcgt cttgcggtaa aggcggagac gcggcgcagg gcgggcagcc tgctggtcgg      120gaagcccctg cgcccgtcgt cggtgtcgta accgtccatc cgcaaaccgt cgcattgacc      180gtcgagttgc cggggcgttt ggaatcgctg cgtaccgccg atgtccgcgc ccaagtcggc      240ggcatcatcc aaaaacgcct gttccaagaa ggcagttatg tccgtgccgg acagccgctg      300tatcagatcg acagttccac ttatgaagca aatctggaaa gcgcgcgcgc gcaactggca      360acggctcagg caacgcttgc caaagcggat gcggatttgg cgcgatacaa gcctttggtt 420gccgccgaag ccgtcagccg gcaggaatac gatgctgcgg taacggcgaa acgttctgcc      480gaggcaggtg tcaaagcagc acaggcggca atcaaatctg ccggcattaa tctgaaccgt      540tcgcgcatta ccgcgccgat ttccggcttt atcggtcagt ccaaagtttc cgaaggtacg      600ctgttgaatg cgggcgatac gaccgtgctg gcaaccatcc gccaaaccaa tccgatgtat      660gtgaacgtta cccagtctgc atccgaagtg atgaaattgc gccgtcagat agccgaaggc      720aaactgctgg cggcggatgg tgtgattgcg gtcggcatca aatttgacga cggcacagtt      780taccctgaaa aaggccgcct gctgtttgcc gatccggtcg tcaacgaatc gaccggtcag      840attaccctgc gcgccgccgt accgaacgat cagaatatcc tgatgcccgg tctgtatgtg      900cgcgtgctga tggaccaagt ggcggtggat aacgcatttg ttgtgccgca gcaggcggta      960acgcgcggtg cgaaagatac cgtgatgatt gtgaatgccc aaggcggtat ggaaccccgc     1020gaggtaacgg ttgcgcaaca gcagggtacg aattggattg ttacgtcggg tctgaaggac     1080ggggacaagg tggttgtgga aggcatcagt atcgccggta taacgggtgc gaaaaaggta     1140acgcccaaag aatgggcgtc gtctgaaaac caagccgccg cgcctcaatc cggcgttcag     1200acggcatctg aagccaaaac tgcttctgaa gcggaataa                            1239<210>2<211>412<212>PRT<213>脑膜炎Neisseria Meningitidis <400> 2MET Ala PHE TYR Ala PHE LYS Ala Met Ala Ala Ala Ala Ala Ala Ala Val Ala Leu Val Leu Ser Cyl LYS GLY GLY ASP ALA ALA ALA ALA ALA ALA ALA ALA ALALA AL AL AL AL AL AS ’s Pro Ala Gly Arg Glu Ala Pro Ala Pro Val Val Gly35                  40                  45Val Val Thr Val His Pro Gln Thr Val Ala Leu Thr Val Glu Leu Pro50                  55                  60Gly Arg Leu Glu Ser Leu Arg Thr Ala Asp Val Arg Ala Gln Val Gly65                  70                  75                  80Gly Ile Ile Gln Lys Arg Leu Phe Gln Glu Gly Ser Tyr Val Arg Ala85                  90                  95Gly Gln Pro Leu Tyr Gln Ile Asp Ser Ser Thr Tyr Glu Ala Asn Leu100                 105                 110Glu Ser Ala Arg Ala Gln Leu Ala Thr Ala Gln Ala Thr Leu Ala Lys115                 120                 125Ala Asp Ala Asp Leu Ala Arg Tyr Lys Pro Leu Val Ala Ala Glu Ala130                 135                 140Val Ser Arg Gln Glu Tyr Asp Ala Ala Val Thr Ala Lys Arg Ser Ala145                 150                 155                 160Glu Ala Gly Val Lys Ala Ala Gln Ala Ala Ile Lys Ser Ala Gly Ile165 170                 175Asn Leu Asn Arg Ser Arg Ile Thr Ala Pro Ile Ser Gly Phe Ile Gly180                 185                 190Gln Ser Lys Val Ser Glu Gly Thr Leu Leu Asn Ala Gly Asp Thr Thr195                 200                 205Val Leu Ala Thr Ile Arg Gln Thr Asn Pro Met Tyr Val Asn Val Thr210                 215                 220Gln Ser Ala Ser Glu Val Met Lys Leu Arg Arg Gln Ile Ala Glu Gly225                 230                 235                 240Lys Leu Leu Ala Ala Asp Gly Val Ile Ala Val Gly Ile Lys Phe Asp245                 250                 255Asp Gly Thr Val Tyr Pro Glu Lys G1y Arg Leu Leu Phe Ala Asp Pro260                 265                 270Val Val Asn Glu Ser Thr Gly Gln Ile Thr Leu Arg Ala Ala Val Pro275                 280                 285Asn Asp Gln Asn Ile Leu Met Pro Gly Leu Tyr Val Arg Val Leu Met290                 295                 300Asp Gln Val Ala Val Asp Asn Ala Phe Val Val Pro Gln Gln Ala Val305                 310                 315                 320Thr Arg Gly Ala Lys Asp Thr Val Met Ile Val Asn Ala Gln Gly Gly325                 330                 335Met Glu Pro Arg Glu Val Thr Val Ala Gln Gln Gln Gly Thr Asn Trp340                 345                 350Ile Val Thr Ser Gly Leu Lys Asp Gly Asp Lys Val Val Val Glu Gly355                 360                 365Ile Ser Ile Ala Gly Ile Thr Gly Ala Lys Lys Val Thr Pro Lys Glu370                 375                 380Trp Ala Ser Ser Glu Asn Gln Ala Ala Ala Pro Gln Ser Gly Val Gln385                 390                 395                 400Thr Ala Ser Glu Ala Lys Thr Ala Ser Glu Ala Glu405 410<210>3<211>41<212>DNA<213>Artificial sequence<220><223>Primer<400>3aggcagaggc atatggcttt ttatgctttt aaggcgat g<21 2>4 DNA <213>artificial sequence<220><223>primer<400>4aggcagaggc tcgagttccg cttcagaagc agttttggct tc 42

Claims (18)

CLAIMS 1. An isolated polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 2. 2. The isolated polypeptide of claim 1, wherein the amino acid sequence has at least 95% identity to the amino acid sequence of SEQ ID NO:2. 3. The isolated polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:2. 4. The isolated polypeptide of SEQ ID NO:2. 5. The immunogenic fragment of the polypeptide of any one of claims 1-4, wherein the immunogenic activity of the immunogenic fragment is substantially identical to the polypeptide of SEQ ID NO:2. 6. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:2. 7. An isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 1. 8. An isolated polynucleotide containing a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2, which can be selected for suitable by using a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof under stringent hybridization conditions Library acquisition. 9. An expression vector or a recombinant living microorganism comprising the isolated polynucleotide of any one of claims 6-8. 10. The method for expressing the polynucleotide according to any one of claims 6-8, comprising transforming a host cell with an expression vector containing at least one said polynucleotide, and under conditions sufficient to express any one of said polynucleotide The host cells were cultured. 11. A vaccine composition comprising an effective amount of a polypeptide according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier. 12. A vaccine composition comprising an effective amount of a polynucleotide according to any one of claims 6 to 8 and a pharmaceutically acceptable carrier. 13. The vaccine composition according to any one of claims 11 or 12, wherein said composition contains at least one additional Neisseria meningitidis antigen. 14. An antibody immunospecific for the polypeptide or immunological fragment of any one of claims 1 to 5. 15. A method of diagnosing a Neisseria meningitidis infection comprising identifying the polypeptide of any one of claims 1-5 present in a biological sample from an animal suspected of having such an infection, or having immunospecificity for said polypeptide antibodies. 16. Use of a composition comprising an immunologically effective amount of a polypeptide according to any one of claims 1-5 for the manufacture of a medicament for generating an immune response in an animal. 17. Use of a composition comprising an immunologically effective amount of a polynucleotide according to any one of claims 6-8 for the manufacture of a medicament for generating an immune response in an animal. 18. A therapeutic composition for the treatment of humans suffering from Neisseria meningitidis disease comprising at least one antibody against the polypeptide of claims 1-5 together with a suitable pharmaceutically acceptable carrier.

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